Plants with reduced activity of a class 3 branching enzyme

ABSTRACT

The present invention relates to plant cells and plants, which are genetically modified, wherein the genetic modification leads to the reduction of the activity of a Class 3 vegetable branching enzyme in comparison with corresponding wild type plant cells or wild type plants that have not been genetically modified. Furthermore, the present invention relates to means and methods for the manufacture of such plant cells and plants. Plant cells and plants of this type synthesise a modified starch. The present invention therefore also relates to the starch synthesised by the plant cells and plants according to the invention as well as to methods for the manufacture of the starch and to the manufacture of starch derivatives of this starch. Furthermore, the present invention relates to nucleic acids coding a Class 3 branching enzyme, vectors, host cells, plant cells and plants containing such nucleic acid molecules.

The present invention relates to plant cells and plants, which aregenetically modified, wherein the genetic modification leads to thereduction of the activity of a Class 3 vegetable branching enzyme incomparison with corresponding wild type plant cells or wild type plantsthat have not been genetically modified. Furthermore, the presentinvention relates to means and methods for the manufacture of such plantcells and plants. Plant cells and plants of this type synthesise amodified starch. The present invention therefore also relates to thestarch synthesised by the plant cells and plants according to theinvention as well as to methods for the manufacture of the starch and tothe manufacture of starch derivatives of this starch. Furthermore, thepresent invention relates to nucleic acids coding a Class 3 branchingenzyme, vectors, host cells, plant cells and plants containing suchnucleic acid molecules.

With regard to the increasing importance currently attributed tovegetable constituents as renewable raw material sources, one of thetasks of biotechnological research is to endeavour to adapt thesevegetable raw materials to suit the requirements of the processingindustry. Furthermore, in order to enable regenerating raw materials tobe used in as many areas of application as possible, it is necessary toachieve a large variety of materials.

Polysaccharide starch is made up of chemically uniform base components,the glucose molecules, but constitutes a complex mixture of differentmolecule forms, which exhibit differences with regard to the degree ofpolymerisation and branching, and therefore differ strongly from oneanother in their physical-chemical characteristics. Discrimination ismade between amylose starch, an essentially unbranched polymer made fromα-1,4-glycosidically linked glucose units, and the amylopectin starch, abranched polymer, in which the branches come about by the occurrence ofadditional α-1,6-glycosidic links. A further essential differencebetween amylose and amylopectin lies in the molecular weight. Whileamylose, depending on the origin of the starch, has a molecular weightof 5×10⁵-10⁶ Da, that of the amylopectin lies between 10⁷ and 10⁸ Da.The two macromolecules can be differentiated by their molecular weightand their different physical-chemical characteristics, which can mosteasily be made visible by their different iodine bondingcharacteristics. Amylose has long been looked upon as a linear polymer,consisting of α-1,4-glycosidically linked α-D-glucose monomers. In morerecent studies, however, the presence of α-1,6-glycosidic branchingpoints (ca. 0.1%) has been shown (Hizukuri and Takagi, Carbohydr. Res.134, (1984), 1-10; Takeda et al., Carbohydr. Res. 132, (1984), 83-92).

Amylopectin constitutes a complex mixture of differently branchedglucose chains. In contrast to amylose, amylopectin is more stronglybranched. According to textbook information (Voet and Voet,Biochemistry, John Wiley & Sons, 1990), on average, the α-1,6 branchesoccur every 24 to 30 glucose residues. This is equivalent to a degree ofbranching of ca. 3%-4%. The figures for the degree of branching arevariable and are dependent on the origin (e.g. plant species, plant typeetc.) of the appropriate starch. In typical plants used for theindustrial production of starch, such as maize, wheat or potato, forexample, the synthesised starch consists of ca. 20%-30% amylose starchand ca. 70%-80% amylopectin starch.

The functional characteristics of the starch, along with theamylose/amylopectin ratio and the phosphate content, are stronglyaffected by the molecular weight, the pattern of the side chaindistribution, the ion concentration, the lipid and protein content, theaverage grain size of the starch and the grain morphology of the starchetc. At the same time, by way of example, the solubility, theretrogradation behaviour, the water bonding capability, the filmformation characteristics, the viscosity, the sticking characteristics,the freezing-thawing stability, the acid stability, the gelling strengthetc. must be mentioned as important functional characteristics. Thegrain size of the starch can also be important for differentapplications.

Branching enzymes, which are also abbreviated by the designation “BE”(from Branching Enzyme; E. C. 2.4.1.18), catalyse the introduction ofα-1,6 branches in α-1,4-glucans. Branching enzymes and the nucleic oramino acid sequences that characterise them are known from widelydifferent organisms, such as bacteria, microbial fungi, mammals, algaeand higher plants, for example. As only plants synthesise starch, whilethe above-mentioned non-vegetable organisms (e.g. bacteria, fungi andmammals) synthesise glycogen, the related branching enzymes, which areinvolved in the synthesis of the appropriate polymer, can also besub-divided into glycogen branching enzymes and starch branchingenzymes. Plants are therefore starch branching enzymes, which are oftenalso referred to as Q-enzymes in older literature.

In all plant species that have been investigated up to now, thebranching enzymes described can be associated with two different classes(Burton et al., 1995, Plant Journal 7, 3-15; Mizuno et al., 2001, PlantCell Physiol. 42(4), 349-357). The association with these classes,sometimes designated in the literature with A or 2 respectively and B or1 respectively, is based on the comparison of derived protein sequences.

As different nomenclatures have been used in the past for designatingand classifying branching enzymes, Smith-White and Preiss (1994, PlantMolecular Biology Reporter 12, 67-71) (1994, Plant Molecular BiologyReporter 12, 67-71) have proposed a system for standardising thisnomenclature, in which the association with the two classes of vegetablebranching enzymes is also based on the comparison of derived proteinsequences (Larsson et al., 1998, Plant Mol. Biol. 37, 505-511).According to this nomenclature, those vegetable branching enzymes, theamino acid sequence of which has a higher degree of identity with thatof branching enzyme I of maize (GenBank Acc: D11081), is to bedesignated as a Class 1 branching enzyme, and those vegetable branchingenzymes, the coding amino acid sequence of which has a higher degree ofidentity with that of branching enzyme II of maize (GenBank Acc:AF072725), is to be designated as a Class 2 branching enzyme. Thedesignation of gene products, which are coding for branching enzymes,are, in accordance with the nomenclature of Smith-White and Preiss, tobe incorporated in the already existing nomenclature by means of E.C.numbers. This results in the so-called GPN (Gene Product Number) Codesfor the two classes, namely GPN 2.2.4.1.18:1 for Class 1 branchingenzymes and GPN 2.2.4.18:2 for Class 2 branching enzymes.

The following vegetable or starch branching enzymes therefore belong toClass 1 (GPN 2.2.1.18:1) according to the nomenclature proposed bySmith-White and Preiss (1994, Plant Molecular Biology Reporter 12,67-71):

BE I from Aegilops tauschii (GenBank Acc: AF525746), BE I from barley(GenBank Acc: AY304541), BE from tapioca (GenBank Acc: X77012), BE I(frequently also described as BE 1) from rice (GenBank Acc: D11082,D10752, D10838), BE 3 from bean (GenBank Acc: AB029549), BE II from pea(GenBank Acc: X80010), BE from millet (GenBank Acc: AF169833), BE I frompotato (GenBank Acc: Y08786, X69805), BE from wheat (GenBank Acc:Y12320, AF076679, AF002820) and BE I from maize (GenBank Acc: D11081,AAO20100, E03435, AY176762, U17897, AF072724).

At the same time, the amino acid sequences for different Class 1branching enzymes each have an identity of more than 60% with the aminoacid sequence of branching enzyme I from maize (GenBank Acc: D11081).

Branching enzymes, which belong to Class 2 (GPN 2.2.1.18:2) according tothe nomenclature proposed by Smith-White and Preiss (1994, PlantMolecular Biology Reporter 12, 67-71) are, for example, BE IIa fromAegilops tauschii (GenBank Acc: AF338431, WO 9914314), BE2-1 and BE2-2from Arabidosis thaliana (BE2-1 GenBank Acc: NM_(—)129196 CAA04134;BE2-2 GenBank Acc: CAB82930, NM_(—)120446), BE IIa and BE IIb frombarley (BE IIa GenBank Acc: AF064560; BE IIb GenBank Acc: AF064561), BEII from sweet potato (GenBank Acc: AB071286), BE III and BE IV(frequently also described as BE 3 or BE 4 respectively) from rice (BEiii GenBank Acc: D16201; BE IV GenBank Acc: AB023498), BE 1 from bean(GenBank Acc: AB029548), BE I from pea (GenBank Acc: X80009), BE IIbfrom millet (GenBank Acc: AY304540), BE II from potato (GenBank Acc:AJ000004, AJ011885, AJ011888, AJ011889, AJ011890), BE II or BE ha fromwheat (GenBank Acc: Y11282, AF286319, AF338432, U66376) and BE II, or BEIIb from maize (BE II GenBank Acc: AAA18571, T02981; BE IIb GenBank Acc:AF072725, L08065). At the same time, the amino acid sequences fordifferent Class 2 branching enzymes each have an identity of more than60% with the amino acid sequence of branching enzyme IIb from maize(GenBank Acc: AF072725).

Vegetable or starch branching enzymes belong to the family ofalpha-amylolytic enzymes (Svensson, 1994, Plant Molecular Biology 25,141-157; Jespersen et al., 1991, Biochem J. 280, 51-55) and, with regardto the amino acid sequence, have four conserved domains (Baba et al.,1991, Biochem. Biophys. Res. Commun. 181(1), 87-94; Kuriki et al., 1996,J. of Protein Chemistry 15(3), 305-313).

Structural predictions based on mathematical calculations derived fromexperimental data such as protein crystal structures (Pfam:http://hits.isb-sib.ch/cgi-bin/PFSCAN?) show that all previously knownbranching enzymes from higher plants have two domains: an alpha-amylasedomain and an iso-amylase domain. Here, the iso-amylase domain liescloser to the N-terminus of the protein than the alpha-amylase domain.

Plants are known, for example, which have a reduced activity of a Class2 branching enzyme due to a mutation. These include the so-called“amylose extender” (ae) mutants from maize (Stindard et al., 1993, PlantCell 5, 1555-1566; Boyer and Preiss, 1978, Biochem. Biophys. Res.Commun. 80,169-175) and rice (Mizuno et al., 1993, J. Biol. Chem. 268,19084-19091), as well as the “rugosus” (r) mutation in pea (Smith, 1988,Planta 175, 270-279; Bhattacharyya et al., 1990, Cell 60, 115-122). Allthese mutants are distinguished by the fact that they synthesise astarch, which has an increased amylose content in comparison withstarches from corresponding plants, which do not have this mutation.

Furthermore, genetically modified potato plants are described, in whichthe activity of a BE I (Class 1) branching enzyme (Kossmann et al.,1991, Mol Gen Genet 230, 39-4; Safford et al., 1998, CarbohydratePolymers 35, 155-168), or the activity of a BEII (Class 2) branchingenzyme (Jobling et al., 1999, The Plant Journal 18), or the activity ofa BEI and BEII branching enzyme (Schwall et al., 2000, NatureBiotechnology 18, 551-554, Jobling et al., 2003, Nature Biotechnology21, 77-80) are reduced.

Previously, it has been possible to associate all vegetable branchingenzymes to one or both of the classes described above. Plant cells orplants, which have a reduced activity of a branching enzyme, whichcannot be associated with these classes, are unknown.

The object of the present invention is therefore based on providingmodified starches, new plant cells and/or plants, which synthesise sucha modified starch, as well as means and methods for producing saidplants.

This problem is solved by the embodiments described in the claims.

The present invention therefore relates to genetically modified plantcells and genetically modified plants, characterised in that the plantcells or plants have a reduced activity of at least one Class 3branching enzyme in comparison with corresponding wild type plant cellsor wild type plants that have not been genetically modified.

A first aspect of the present invention relates to a plant cell orplant, which is genetically modified, wherein the genetic modificationleads to the reduction of the activity of at least one Class 3 branchingenzyme in comparison with corresponding wild type plant cells or wildtype plants that have not been genetically modified.

At the same time, the genetic modification can be any geneticmodification, which leads to a reduction of the activity of at least oneClass 3 branching enzyme in comparison with corresponding wild typeplant cells or wild type plants that have not been genetically modified.

In conjunction with the present invention, the term “wild type plantcell” means that the plant cells concerned were used as startingmaterial for the manufacture of the plant cells according to theinvention, i.e. their genetic information, apart from the introducedgenetic modification, corresponds to that of a plant cell according tothe invention.

In conjunction with the present invention, the term “wild type plant”means that the plants concerned were used as starting material for themanufacture of the plants according to the invention, i.e. their geneticinformation, apart from the introduced genetic modification, correspondsto that of a plant according to the invention.

In conjunction with the present invention, the term “corresponding”means that, in the comparison of several objects, the objects concernedthat are compared with one another have been kept under the sameconditions. In conjunction with the present invention, the term“corresponding” in conjunction with wild type plant cell or wild typeplant means that the plant cells or plants, which are compared with oneanother, have been raised under the same cultivation conditions and thatthey have the same (cultivation) age.

In an embodiment of the present invention, the genetic modification ofthe plant cells according to the invention or the plants according tothe invention is brought about by mutagenesis of one or more genes. Thetype of mutation is not important, as long as it leads to a reduction inthe activity of a Class 3 branching enzyme.

In conjunction with the present invention, the term “mutagenesis” is tobe understood to mean any type of introduced mutation, such asdeletions, point mutations (nucleotide exchanges), insertions,inversions, gene conversions or chromosome translocations, for example.

Here, the mutation, which leads to the reduction of the activity of atleast one endogenous Class 3 branching enzyme, can be produced by theuse of chemical agencies or energy-rich radiation (e.g. x-rays, neutronradiation, gamma radiation or UV radiation).

Agencies, which can be used to produce chemically induced mutations, andthe mutations resulting from the effect of the corresponding mutagensare, for example described in Ehrenberg and Husain, 1981, (MutationResearch 86, 1-113), Müller, 1972 (Biologisches Zentralblatt 91 (1),31-48). The production of rice mutants using gamma radiation, ethylmethane sulphonate (EMS), N-methyl-N-nitrosurea or sodium azide (NaN₃)is described, for example, in Jauhar and Siddiq (1999, Indian Journal ofGenetics, 59 (1), 23-28), in Rao (1977, Cytologica 42, 443-450), Guptaand Sharma (1990, Oryza 27, 217-219) and Satoh and Omura (1981, JapaneseJournal of Breeding 31 (3), 316-326). The production of wheat mutantsusing NaN₃ or maleic hydrazide is described in Arora et al. (1992,Annals of Biology 8 (1), 65-69). An overview of the production of wheatmutants using different types of energy-rich radiation and chemicalagencies is presented in Scarascia-Mugnozza et al. (1993, MutationBreeding Review 10, 1-28). Svec et al. (1998, Cereal ResearchCommunications 26 (4), 391-396) describes the use ofN-ethyl-N-nitrosurea for producing mutants in triticals. The use of MMS(methyl methane sulphonic acid) and gamma radiation for the productionof millet mutants is described in Shashidhara et al. (1990, Journal ofMaharashtra Agricultural Universities 15 (1), 20-23).

The manufacture of mutants in plant species, which mainly propagatevegetatively, has been described, for example, for potatoes, whichproduce a modified starch (Hovenkamp-Hermelink et al. (1987, Theoreticaland Applied Genetics 75, 217-221) and for mint with increased oil yieldor modified oil quality (Dwivedi et al., 2000, Journal of Medicinal andAromatic Plant Sciences 22, 460-463).

All these methods are basically suitable for manufacturing the plantcells according to the invention and the plants according to theinvention.

Mutations in the appropriate genes, in particular in genes coding aClass 3 branching enzyme, can be found with the help of methods known tothe person skilled in the art. In particular, analyses based onhybridisations with probes (Southern Blot), amplification by means ofpolymerase chain reaction (PCR), sequencing of related genomic sequencesand the search for individual nucleotide exchanges can be used for thispurpose. A method of identifying mutations based on hybridisationpatterns is, for example, the search for restriction fragment lengthdifferences (Restriction Fragment Length Polymorphism, RFLP) (Nam etal., 1989, The Plant Cell 1, 699-705; Leister and Dean, 1993, The PlantJournal 4 (4), 745-750). A method based on PCR is, for example, theanalysis of amplified fragment length differences (Amplified FragmentLength Polymorphism, AFLP) (Castiglioni et al., 1998, Genetics 149,2039-2056; Meksem et al., 2001, Molecular Genetics and Genomics 265,207-214; Meyer et al., 1998, Molecular and General Genetics 259,150-160). The use of amplified fragments cut with restrictionendonucleases (Cleaved Amplified Polymorphic Sequences, CAPS) can becalled upon for the identification of mutations (Konieczny and Ausubel,1993, The Plant Journal 4, 403-410; Jarvis et al., 1994, Plant MolecularBiology 24, 685-687; Bachem et al., 1996, The Plant Journal 9 (5),745-753). Methods for the determination of SNPs have been described byQi et al. (2001, Nucleic Acids Research 29 (22), e116) Drenkard et al.(2000, Plant Physiology 124, 1483-1492) and Cho et al. (1999, NatureGenetics 23, 203-207) amongst others. Methods, which allow severalplants to be investigated for mutations in certain genes in a shorttime, are particularly suitable. Such a method, so-called TILLING(Targeting Induced Local Lesions IN Genomes), has been described byMcCallum et al. (2000, Plant Physiology 123, 439-442).

These methods are basically suitable for identifying plant cellsaccording to the invention and plants according to the invention.

Hoogkamp et al. (2000, Potato Research 43, 179-189) have manufacturedstable monoploid mutants starting from a potato mutant (amf), which wasmanufactured by means of chemical mutagens. These plants do notsynthesise any more active enzyme for a starch synthesis connected tothe starch grain (GBSS I) and therefore produce an amylose-free starch.The monoploid potato plants obtained can be used as starting materialfor further mutageneses in order to identify plants, which synthesise astarch with modified characteristics. The plant cells according to theinvention and plants according to the invention, which produce a starchaccording to the invention, can be identified and isolated byappropriate methods.

The plant cells according to the invention and the plants according tothe invention have a reduction of the activity of at least one Class 3branching enzyme in comparison with corresponding wild type plant cellsthat have not been genetically modified.

Here, within the framework of the present invention, the term “reductionof activity” means a reduction in the expression of endogenous genes,which code Class 3 branching enzymes, and/or a reduction in the quantityof protein of a Class 3 branching enzyme in the plant cells and/or areduction in the enzymatic activity of Class 3 branching enzymes in theplant cells.

The reduction in the expression can, for example, be determined bymeasuring the quantity of transcripts coding Class 3 branching enzyme,e.g. using Northern blot analysis or RT-PCR. Here, a reductionpreferably means a reduction in the amount of transcripts in comparisonwith corresponding plant cells that have not been genetically modifiedby at least 50%, in particular by at least 70%, preferably by at least85% and particularly preferably by at least 95%.

The reduction in the amount of protein of a Class 3 branching enzyme,which results in a reduced activity of this protein in the plant cellsconcerned, can, for example, be determined by immunological methods suchas Western blot analysis, ELISA (Enzyme Linked Immuno Sorbent Assay) orRIA (Radio Immune Assay). Here, a reduction preferably means a reductionin the amount of Class 3 branching enzyme protein in comparison withcorresponding plant cells that have not been genetically modified by atleast 50%, in particular by at least 70%, preferably by at least 85% andparticularly preferably by at least 95%. Within the framework of thepresent invention, the term “branching enzyme” (α-1,4-glucan:α-1,4-glucan 6-glycosyltransferase, E.C. 2.4.1.18) is understood to meana protein, which catalyses a transglycosylation reaction, in which α-1,4links of an α-1,4-glucan donor are hydrolysed and the thereby releasedα-1,4-glucan chains are transferred to an α-1,4-glucan acceptor chainand, in doing so, are transformed into α-1,6-links. In particular,within the framework of the present invention, the term “branchingenzyme” is to be understood to mean a vegetable branching enzyme, i.e. astarch branching enzyme.

The activity of a branching enzyme can be demonstrated, for example,with the help of native acrylamide gel electrophoresis. In doing so,proteins are first separated electrophoretically and, after incubationin buffers containing an activity, which synthesises linear α-1,4-glucanchains (e.g. starch phosphorylase a) and its substrate (e.g.glucose-6-phosphate), the corresponding gels are coloured with iodine(Kimihiko et al., 1980, Analytical Biochemistry 108, 16-24).

Furthermore, branching enzymes in microbial organisms, such as the E.coli strain KV832 for example (Kiel et al., 1987 Mol. Gen. Genet 207:294-301), which do not synthesise branched α-glucans, can be expressed.If an activity of a branching enzyme is introduced into the microbialorganism due to the expression of a foreign gene in such strains (e.g.E. coli KV832), then the branching enzyme activity can be demonstratedby treating colonies of these organisms with iodine vapour, for example.Colonies, which synthesise linear α-1,4-glucans, turn blue in this test,while colonies, which synthesise branched glucans by expressing anadditional enzymatic activity of a branching enzyme, turn reddish-brownafter treating with iodine vapour. It is also possible to expressproteins in phosphoglucomutase mutants of E. coli to identify abranching enzyme activity of appropriate proteins (Buettcher et al.,1999, Biochem. Biophys. Acta 1432, 406-412).

A further possibility of demonstrating branching enzyme activity ofproteins is the use of a reaction stimulated by phosphorylase a and thesubsequent separation of the products by means of thin filmchromatography (Almstrupp et al., 2000, Analytical Biochemistry 286,297-300).

Branching enzyme activities can also be demonstrated with the help ofthe methods described in Guan and Preiss 11993, Plant. Physiol. 102,1269-1273) and Kuriki et al. (1996, J. of Protein Chemistry 15,305-313).

In conjunction with the present invention, the term “Class 3 branchingenzyme” is to be understood as a branching enzyme, which has a higherdegree of identity with the amino acid sequence shown in SEQ ID NO 4than with that of the branching enzyme BE I from maize (GenBank Acc:D11081) or with that of the branching enzyme BE IIb from maize (GenBankAcc: AF072725). Preferably, the Class 3 branching enzyme comes fromstarch-storing plants, particularly preferably from plant species of thegenus Solanum, especially preferably from Solanum tuberosum.

In a further embodiment of the present invention, amino acid sequencescoding Class 3 branching enzymes have an identity of at least 60% withthe sequence shown in SEQ ID NO 4, in particular of at least 70%,preferably of at least 80% and particularly preferably of at least 90%and especially preferably of at least 95%.

According to the invention, Class 3 branching enzymes have aniso-amylase domain (Pfam acc.: Pf02922) and an alpha-amylase domain(Pfam acc: Pf00128). According to the invention, the iso-amylase domainand the alpha-amylase domain in amino acid sequences coding branchingenzymes are separated from one another by the presence of further aminoacids, which do not belong to these two domains. Class 3 branchingenzymes according to the invention are distinguished by the fact thatthe iso-amylase domain is separated from the alpha-amylase domain by agreater number of amino acids than the iso-amylase domain and thealpha-amylase domain of Class 1 and 2 branching enzymes.

Class 3 branching enzymes according to the invention are preferablydistinguished with regard to their amino acid sequence by the fact thatthey have at least 70, preferably at least 100, particularly preferablyat least 130 and especially preferably at least 198 amino acids betweenthe iso-amylase domain and the alpha-amylase domain. In a furtherembodiment of the present invention, in the case of an amino acidsequence coding a Class 3 branching enzyme, the C-terminal end of theiso-amylase domain is separated from the N-terminal beginning of thealpha-amylase domain by 70 to 198, preferably by 100 to 198,particularly preferably by 130 to 198 and especially particularlypreferably by 150 to 198 amino acids.

With the help of the Pfam database (Batemann et al., 2002, Nucleic AcidsResearch 30, 276-280; accessible viahttp://www.sanger.ac.uk/Software/Pfam/, http://www.cgb.ki.se/Pfam/;http://pfam.jouy.inra.fr/ or http://pfam.wustl.edu/), it is possible forthe person skilled in the art to determine whether amino acid sequencesalready have known domains (e.g. an iso-amylase domain and/or analpha-amylase domain).

Pfam is a database put together by experts, which classifies amino acidsequences into so-called families. Here, the assignment of an amino acidsequence to a family is carried out on the basis of so-called domains,which are to be looked upon as functional and structural components ofproteins. A domain is defined as a structural unit or a repeatedlyoccurring amino acid sequence unit, which can occur in proteins withwidely different functions. Along with information relating to the aminoacid sequence of known proteins, further knowledge (e.g. evidence of theenzymatic activity, crystal structure data) is also used for theassignment of a protein to a family. Each family is assigned a name andan “accession” number (e.g. Name: Isoamylase_N, acc: PF02922). Aconstituent part of each family in the Pfam database is, amongst otherthings, a so-called “seed alignment”. The “seed alignment” contains theamino acid sequences of representative proteins of a family. Startingfrom “seed alignments”, a so-called profile HMM (“profile Hidden MarkovModel”; overview article in: Durbin et al., “Biological SequenceAnalysis: Probabilistic Models of Proteins and Nucleic Acids”, CambridgeUniversity Press, 1998, ISBN 0-521-62041-4) is produced using the HMMER2 software (freely available under http://hmmer.wustl.edu/). The HMMsproduced have names and are stored in the Pfam database specifically forthe correspondingly assigned domains. In contrast to classical, multiple“alignments” (e.g. produced using the Clustal W program or the Blossum62algorithm), HMMs are based on a valid statistical theory (Bayes theoryof conditional probability, Markoff chains) and enable an interrogationsequence (query) to be assigned to a family based on the use ofposition-specific evaluation matrices. This enables an assignment to bemade even when there are considerable differences in the amino acidsequences between the interrogation sequence (query) and a comparisonsequence (e.g. amino acid sequence entry in a database).

The domain structure of the amino acid sequence concerned can bedetermined by means of a comparison of the HMMs stored in the Pfamdatabase with amino acid sequences, which are entered as a so-calledinterrogation sequence (query) (e.g. underhttp://hits.isb-sib.ch/cgi-bin/PFSCAN?).

In conjunction with the present invention, the term “iso-amylase domain”is to be understood to mean a Pfam iso-amylase domain (acc: Pf02922). Atthe same time, the HMM describing this Pfam iso-amylase domain is to beproduced with the HMMER 2 [2.3.1] software, starting from a “seedalignment”, which contains the amino acid sequences shown in Table 1. Inconjunction with the present invention, the “seed alignment” is producedby means of the ClustalW program (Thompson et al., Nucleic AcidsResearch 22 (1994), 46734680; see below). The following settings must bechosen to produce the appropriate HMMs: Build Method of HMM: hmmbuild—FHMM_Is, hmmcalibrate—seed 0 HMM_Is; Gathering cutoff: 2.3 2.3; Trustedcutoff: 2.3 2.2; Noise cutoff: 2.1 2.1). Further information forproducing the HMM of the Pfam iso-amylase domain (acc: Pf02922) is givenin Table 3.

In conjunction with the present invention, the term “alpha-amylasedomain” is to be understood to mean a Pfam alpha-amylase domain (acc:Pf00128). At the same time, the HMM describing this Pfam alpha-amylasedomain is to be produced with the HMMER 2 [2.3.1] software, startingfrom a “seed alignment”, which contains the amino acid sequences shownin Table 2. Here, the “seed alignment” is produced by means ofHMM_simulated_annealing(http://www.psc.edu/general/software/packages/hmmer/manual/node11.html#SECTION00321000000000000000). The following settings must be chosen toproduce the appropriate HMM: Build Method of HMM: hmmbuild—F HMM_Is,hmmcalibrate—seed 0 HMM_Is; Gathering cutoff: −82.0-82.0; Trustedcutoff: −81.7-81.7; Noise cutoff: −82.7-82.7). Further information forproducing the HMM of the Pfam alpha-amylase domain (acc: Pf00128) isgiven in Table 4.

In conjunction with the present invention, the term “Class 3 branchingenzyme gene” is to be understood to mean a nucleic acid molecule (cDNA,DNA), which codes a Class 3 branching enzyme, preferably a Class 3branching enzyme from starch-storing-plants, particularly preferablyfrom plant species of the genus Solanum, especially preferably fromSolanum tuberosum.

A preferred embodiment of the present invention relates to a geneticallymodified plant cell according to the invention or a genetically modifiedplant according to the invention, wherein the genetic modificationconsists in the introduction of at least one foreign nucleic acidmolecule into the genome of the plant cell or into the genome of theplant.

In this context, the term “genetic modification” means the introductionof homologous and/or heterologous foreign nucleic acid molecules intothe genome of a plant cell or into the genome of a plant, wherein saidintroduction of these molecules leads to a reduction of the activity ofa Class 3 branching enzyme.

The plant cells according to the invention or plants according to theinvention are modified with regard to their genetic information by theintroduction of a foreign nucleic acid molecule. The presence or theexpression of the foreign nucleic acid molecule leads to a phenotypicchange. Here, “phenotypic” change means preferably a measurable changeof one or more functions of the cells. For example, the geneticallymodified plant cells according to the invention and the geneticallymodified plants according to the invention exhibit a reduction of theactivity of a Class 3 branching enzyme due to the presence or on theexpression of the introduced nucleic acid molecule.

In conjunction with the present invention, the term “foreign nucleicacid molecule” is understood to mean such a molecule that either doesnot occur naturally in the corresponding wild type plant cells that havenot been genetically modified, or that does not occur naturally in theconcrete spatial arrangement in wild type plant cells that have not beengenetically modified, or that is localised at a place in the genome ofthe wild type plant cell at which it does not occur naturally.Preferably, the foreign nucleic acid molecule is a recombinant molecule,which consists of different elements, the combination or specificspatial arrangement of which does not occur naturally in vegetablecells.

In principle, the foreign nucleic acid molecule can be any nucleic acidmolecule, which effects a reduction of the activity of a Class 3branching enzyme in the plant cell or plant.

In conjunction with the present invention, the term “genome” is to beunderstood to mean the totality of the genetic material present in avegetable cell. It is known to the person skilled in the art that, aswell as the cell nucleus, other compartments (e.g. plastids,mitochondrions) also contain genetic material.

In a further embodiment, the plant cells according to the invention andthe plants according to the invention are characterised in that theforeign nucleic acid molecule codes a Class 3 branching enzyme,preferably a Class 3 branching enzyme from starch-storing plants,particularly preferably from plants of a species of the genus Solanum,especially preferably from Solanum tuberosum.

In a particularly preferred embodiment, the foreign nucleic acidmolecule codes a Class 3 branching enzyme with the amino acid sequencespecified in SEQ ID NO 4.

A large number of techniques are available for the introduction of DNAinto a vegetable host cell. These techniques include the transformationof vegetable cells with T-DNA using Agrobacterium tumefaciens orAgrobacterium rhizogenes as the transformation medium, the fusion ofprotoplasts, injection, the electroporation of DNA, the introduction ofDNA by means of the biolistic approach as well as other possibilities.

The use of agrobacteria-mediated transformation of plant cells has beenintensively investigated and adequately described in EP 120516; Hoekema,IN: The Binary Plant Vector System Offsetdrukkerij Kanters B. V.,Alblasserdam (1985), Chapter V; Fraley et al., Crit. Rev. Plant Sci. 4,146 and by An et al. EMBO J. 4, (1985), 277-287. For the transformationof potato, see Rocha-Sosa et al., EMBO J. 8, (1989), 29-33, for example.

The transformation of monocotyledonous plants by means of vectors basedon agrobacterium transformation has also been described (Chan et al.,Plant Mol. Biol. 22, (1993), 491-506; Hiei et al., Plant J. 6, (1994)271-282; Deng et al, Science in China 33, (1990), 28-34; Wilmink et al.,Plant Cell Reports 11, (1992), 76-80; May et al., Bio/Technology 13,(1995), 486-492; Conner and Domisse, Int. J. Plant Sci. 153 (1-992),550-555; Ritchie et al, Transgenic Res. 2, (1993), 252-265). Analternative system to the transformation of monocotyledonous plants istransformation by means of the biolistic approach (Wan and Lemaux, PlantPhysiol. 104, (1994), 37-48; Vasil et al., Bio/Technology 11 (1993),1553-1558; Ritala et al., Plant Mol. Biol. 24, (1994), 317-325; Spenceret al., Theor. Appl. Genet. 79, (1990), 625-631), protoplasttransformation, electroporation of partially permeabilised cells and theintroduction of DNA by means of glass fibres. In particular, thetransformation of maize has been described in the literature many times(cf. e.g. WO95/06128, EP0513849, EP0465875, EP0292435; Fromm et al.,Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2,(1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Morocet al., Theor. Appl. Genet. 80, (1990), 721-726).

The successful transformation of other types of cereal has also alreadybeen described, for example for barley (Wan and Lemaux, see above;Ritala et al., see above; Krens et al., Nature 296, (1982), 72-74) andfor wheat (Nehra et al., Plant J. 5, (1994), 285-297). All the abovemethods are suitable within the framework of the present invention.

Amongst other things, the plant cells according to the invention and theplants according to the invention can be differentiated from wild typeplant cells and wild type plants respectively in that they contain aforeign nucleic acid molecule, which does not occur naturally in wildtype plant cells or wild type plants, or in that such a molecule ispresent integrated at a place in the genome of the plant cell accordingto the invention or in the genome of the plant according to theinvention at which it does not occur in wild type plant cells or wildtype plants, i.e. in a different genomic environment. Furthermore, plantcells according to the invention and plants according to the inventionof this type differ from wild type plant cells and wild type plantsrespectively in that they contain at least one copy of the foreignnucleic acid molecule stably integrated within their genome, possibly inaddition to naturally occurring copies of such a molecule in the wildtype plant cells or wild type plants. If the foreign nucleic acidmolecule(s) introduced into the plant cells according to the inventionor into the plants according to the invention is (are) additional copiesof molecules already occurring naturally in the wild type plant cells orwild type plants respectively, then the plant cells according to theinvention and the plants according to the invention can bedifferentiated from wild type plant cells or wild type plantsrespectively in particular in that this additional copy these additionalcopies is (are) localised at places in the genome at which it does notoccur (or they do not occur) in wild type plant cells or wild typeplants. This can be verified, for example, with the help of a Southernblot analysis.

Furthermore, the plant cells according to the invention and the plantsaccording to the invention can preferably be differentiated from wildtype plant cells or wild type plants respectively by at least one of thefollowing characteristics: If the foreign nucleic acid module that hasbeen introduced is heterologous with respect to the plant cell or plant,then the plant cells according to the invention or plants according tothe invention have transcripts of the introduced nucleic acid molecules.These can be verified, for example, by Northern blot analysis or byRT-PCR (Reverse Transcription Polymerase Chain Reaction). Plant cellsaccording to the invention and plants according to the invention, whichexpress an antisense and/or an RNAi transcript, can be verified, forexample, with the help of specific nucleic acid probes, which arecomplimentary to the RNA (occurring naturally in the plant cell), whichis coding for the protein.

If the foreign nucleic acid module that has been introduced ishomologous with respect to the plant cell or plant, the plant cellsaccording to the invention or plants according to the invention can bedifferentiated from wild type plant cells or wild type plantsrespectively due to the additional expression of the introduced foreignnucleic acid molecule, for example. The plant cells according to theinvention and the plants according to the invention preferably contain(sense and/or antisense) transcripts of the foreign nucleic acidmolecules. This can be demonstrated by Northern blot analysis, forexample, or with the help of so-called quantitative PCR.

In a special embodiment, the plant cells according to the invention andthe plants according to the invention are transgenic plant cells ortransgenic plants respectively.

In a further embodiment, the present invention relates to plant cellsaccording to the invention and plants according to the invention whereinthe foreign nucleic acid molecule is chosen from the group consisting of

-   -   a) Nucleic acid molecules, which code a protein with the amino        acid sequence given under Seq ID NO 4;    -   b) Nucleic acid molecules, which code a protein, the amino acid        sequence of which has an identity of at least 50% with the amino        acid sequence given under SEQ ID NO: 4;    -   c) Nucleic acid molecules, which include the nucleotide sequence        shown under Seq ID NO 3 or a complimentary sequence;    -   d) Nucleic acid molecules, the nucleic acid sequence of which        has an identity of at least 50% with the nucleic acid sequences        described under a) or c);    -   e) Nucleic acid molecules, which hybridise with at least one        strand of the nucleic acid molecules described under a) or c)        under stringent conditions;    -   f) Nucleic acid molecules, the nucleotide sequence of which        deviates from the sequence of the nucleic acid molecules        identified under a), b), c), d), e) or f) due to the        degeneration of the genetic code; and    -   g) Nucleic acid molecules, which represent fragments, allelic        variants and/or derivatives of the nucleic acid molecules        identified under a), b), c), d), e) or f).

A further embodiment of the present invention relates to plant cellsaccording to the invention and plants according to the invention whereinthe foreign nucleic acid molecule is chosen from the group consisting of

-   -   a) Nucleic acid molecules, which code at least one antisense        RNA, which effects a reduction in the expression of at least one        endogenous gene, which codes a Class 3 branching enzyme;    -   b) Nucleic acid molecules, which by means of a co-suppression        effect lead to the reduction in the expression of at least one        endogenous gene, which codes a Class 3 branching enzyme;    -   c) Nucleic acid molecules, which code at least one ribozyme,        which splits specific transcripts of at least one endogenous        gene, which codes a Class 3 branching enzyme;    -   d) Nucleic acid molecules, which simultaneously code at least        one antisense RNA and at least one sense RNA, wherein the said        antisense RNA and the said sense RNA form a double-stranded RNA        molecule, which effects a reduction in the expression of at        least one endogenous gene, which codes a Class 3 branching        enzyme (RNAi technology);    -   e) Nucleic acid molecules introduced by means of in vivo        mutagenesis, which lead to a mutation or an insertion of a        heterologous sequence in at least one endogenous gene coding a        Class 3 branching enzyme, wherein the mutation or insertion        effects a reduction in the expression of a gene coding a Class 3        branching enzyme or results in the synthesis of inactive Class 3        branching enzymes;    -   f) Nucleic acid molecules, which code an antibody, wherein the        antibody results in a reduction in the activity of a Class 3        branching enzyme due to the bonding to a Class 3 branching        enzyme.    -   g) DNA molecules, which contain transposons, wherein the        integration of these transposons leads to a mutation or an        insertion in at least one endogenous gene coding a Class 3        branching enzyme, which effects a reduction in the expression of        at least one gene coding a Class 3 branching enzyme, or results        in the synthesis of inactive Class 3 branching enzymes; and/or    -   h) T-DNA molecules, which, due to insertion in at least one        endogenous gene coding a Class 3 branching enzyme, effect a        reduction in the expression of at least one gene coding a Class        3 branching enzyme, or result in the synthesis of inactive Class        3 branching enzyme.

The plant cells according to the invention and plants according to theinvention can be manufactured by different methods known to the personskilled in the art. These include, for example, the expression of acorresponding antisense RNA or of a double-stranded RNA construct, theprovision of molecules or vectors, which impart a cosuppression effect,the expression of a correspondingly constructed ribozyme that splitsspecific transcripts, which code a Class 3 branching enzyme, orso-called “in vivo mutagenesis”. Furthermore, the reduction of the Class3 branching enzyme activity in plant cells and plants can also bebrought about by the simultaneous expression of sense and antisense RNAmolecules of the respective target gene to be repressed, preferably ofthe Class 3 branching enzyme gene.

In addition to this, it is known that in planta the formation ofdouble-stranded RNA molecules of promoter sequences can lead in trans tomethylation and transcriptional inactivation of homologous copies ofthis promoter (Mette et al., EMBO J. 19, (2000), 5194-5201).

A further possible way in which to reduce the enzymatic activity ofproteins in plant cells or plants is the so-called immunomodulationmethod. It is known that an in planta expression of antibodies, whichspecifically recognise a vegetable protein, results in a reduction ofthe activity of the proteins concerned in appropriate plant cells due tothe formation of a protein antibody complex (Conrad and Manteufel,Trends in Plant Science 6, (2001), 399402; De Jaeger et al., PlantMolecular Biology 43, (2000), 419428; Jobling et al., NatureBiotechnology 21, (2003), 77-80).

All these methods are based on the introduction of a foreign or ofseveral foreign nucleic acid molecules into the genome of plant cells orplants and are therefore basically suitable for manufacturing plantcells according to the invention and plants according to the invention.

For inhibiting the expression of genes by means of antisense orcosuppression technology, a DNA molecule can be used, for example, whichincludes the whole coding sequence for a Class 3 branching enzyme,including any existing flanking sequences, as well as DNA molecules,which include only parts of the coding sequence, whereby these partsmust be long enough to produce an antisense effect or a cosuppressioneffect respectively in the cells. In general, sequences up to a minimumlength of 21 bp, preferably a minimum length of at least 100 bp,particularly preferably of at least 500 bp are suitable. For example,the DNA molecules have a length of 21-100 bp, preferably of 100-500 bp,particularly preferably over 500 bp.

The use of DNA sequences, which have a high degree of identity with theendogenous sequences occurring in the plant cells and which code Class 3branching enzymes, is also suitable for antisense or cosuppressionapproaches. The minimum identity should be greater than ca. 65%,preferably greater than 80%. The use of sequences with identities of atleast 90%, in particular between 95% and 100%, is to be preferred. Themeaning of the term “identity” will be defined elsewhere.

Furthermore, the use of introns, i.e. of non-coding areas of genes,which code for Class 3 branching enzymes, is also conceivable forachieving an antisense or a cosuppression effect.

The use of intron sequences for inhibiting the gene expression of genes,which code for starch biosynthesis proteins, has been described in theinternational patent applications WO97/04112, WO97/04113, WO98/37213,WO98/37214.

The person skilled in the art knows how to achieve an antisense and acosuppression effect. For example, the method of cosuppressioninhibition has been described in Jorgensen (Trends Biotechnol. 8 (1990),340-344), Niebel et al., (Curr. Top. Microbiol. Immunol. 197 (1995),91-103), Flavell et al. (Curr. Top. Microbiol. Immunol. 197 (1995),43-46), Palaqui and Vaucheret (Plant. Mol. Biol. 29 (1995), 149-159),Vaucheret et al., (Mol. Gen. Genet. 248 (1995), 311-317), de Borne etal. (Mol. Gen. Genet. 243 (1994), 613-621).

The expression of ribozymes for reducing the activity of particularenzymes in cells is also known to the person skilled in the art, and isdescribed, for example, in EP-B1 0321201. The expression of ribozymes invegetable cells has been described, for example, in Feyter et al. (Mol.Gen. Genet. 250, (1996), 329-338).

The reduction of the activity of a Class 3 branching enzyme in plantcells according to the invention and plants according to the inventioncan also be brought about by the simultaneous expression of sense andantisense RNA molecules (RNAi technology) of the respective target geneto be repressed, preferably of the Class 3 branching enzyme gene.

This can be achieved, for example, by the use of chimeric constructs,which contain “inverted repeats” of the respective target gene or partsof the target gene. In this case, the generic constructs code for senseand antisense RNA molecules of the respective target gene. Sense andantisense RNA are synthesised simultaneously in planta as an RNAmolecule, wherein sense and antisense RNA are separated from one anotherby a spacer, and are able to form a double-stranded RNA molecule. It hasbeen shown that the introduction of inverted repeat DNA constructs intothe genome of plant cells or plants is a very effective method ofrepressing the genes corresponding to the inverted repeat DNA constructs(Waterhouse et al., Proc. Natl. Acad. Sci. USA 95, (1998), 13959-13964;Wang and Waterhouse, Plant Mol. Biol. 43, (2000), 67-82; Singh et al.,Biochemical Society Transactions Vol. 28 part 6 (2000), 925-927; Liu etal., Biochemical Society Transactions Vol. 28 part 6 (2000), 927-929);Smith et al., (Nature 407, (2000), 319-320; international patentapplication WO99/53050 A1). Sense and antisense sequences of the targetgene or the target genes can also be expressed separately from oneanother by means of similar or different promoters (Nap, J-P et al,6^(th) International Congress of Plant Molecular Biology, Quebec,18th-24th June, 2000; Poster S7-27, Presentation Session S7).

The reduction of the activity of a Class 3 branching enzyme in plantcells according to the invention or plants according to the inventioncan therefore also be achieved by producing double-stranded RNAmolecules. In this regard, “inverted repeats” of DNA molecules of Class3 branching enzyme genes or cDNAs are preferably introduced into thegenome of plants, wherein the DNA molecules (Class 3 branching enzymegene or cDNA or fragments of this gene or cDNA) to be transcribed areunder the control of a promoter, which controls the expression of saidDNA molecules.

In addition to this, it is known that the formation of double-strandedRNA molecules from promoter DNA molecules in plants in trans can lead tomethylation and transcriptional inactivation of homologous copies ofthese promoters, which are to be referred to in the following as targetpromoters (Mette et al., EMBO J. 19, (2000), 5194-5201).

It is therefore possible to reduce the gene expression of a particulartarget gene (e.g. branching enzyme Class 3 gene), which is naturallyunder the control of this target promoter, by deactivating the targetpromoter.

This means that, in this case, the DNA molecules, which include thetarget promoters of the genes to be repressed (target genes), incontrast to the original function of promoters in plants, are not usedas control elements for the expression of genes or cDNAs, but arethemselves used as transcribable DNA molecules.

For the production of double-stranded target promoter RNA molecules inplanta, which can occur there as RNA hairpin molecules, constructs arepreferably used, which contain the “inverted repeats” of the targetpromoter DNA molecules, wherein the target promoter DNA molecules areunder the control of a promoter, which controls the gene expression ofsaid target promoter DNA molecules. These constructs are subsequentlyintroduced into the genome of plants. The expression of the “invertedrepeats” of said target promoter DNA molecules in planta leads to theformation of double-stranded target promoter RNA molecules (Mette etal., EMBO J. 19, (2000), 5194-5201). The target promoter can beinactivated by this means.

The reduction of the activity of a Class 3 branching enzyme in plantcells according to the invention and plants according to the inventioncan therefore also be achieved by the production of double-stranded RNAmolecules of promoter sequences of Class 3 branching enzyme genes. Inthis regard, “inverted repeats” of promoter DNA molecules of Class 3branching enzyme genes are preferably introduced into the genome ofplants, wherein the target promoter DNA molecules (promoter of a Class 3branching enzyme gene) to be transcribed are under the control of apromoter, which controls the expression of said target promoter DNAmolecules.

For inhibiting the expression of genes by means of the simultaneousexpression of sense and antisense RNA molecules (RNAi technology), a DNAmolecule can be used, for example, which includes the whole codingsequence for a Class 3 branching enzyme, including any existing flankingsequences, as well as DNA molecules, which include only parts of thecoding sequence, whereby these parts must be long enough to produce aso-called RNAi effect in the cells. In general, sequences with a minimumlength of 40 bp, preferably a minimum length of at least 100 bp,particularly preferably of at least 500 bp are suitable. For example,the DNA molecules have a length of 21-100 bp, preferably of 100-500 bp.

The use of DNA sequences, which have a high degree of identity with theendogenous sequences occurring in the plant cells and which code Class 3branching enzymes, is also suitable for the simultaneous expression ofsense and antisense RNA molecules (RNAi technology). The minimumidentity should be greater than ca. 65%, preferably greater than 80%.The use of sequences with identities of at least 90%, in particularbetween 95% and 100%, is to be particularly preferred.

Furthermore, the reduction of the activity of a Class 3 branching enzymein plant cells according to the invention and plants according to theinvention can also be achieved by so-called “in vivo mutagenesis”, inwhich a hybrid RNA-DNA oligonucleotide (“Chimeroplast”) is introducedinto plant cells (Kipp, P. B. et al., Poster Session at the “5^(th)International Congress of Plant Molecular Biology, 21st-27th Sep. 1997,Singapore; R. A. Dixon and C. J. Arntzen, meeting report on “MetabolicEngineering in Transgenic Plants”, Keystone Symposia, Copper Mountain,Colo., USA, TIBTECH 15, (1997), 441-447; international patentapplication WO 9515972; Kren et al., Hepatology 25, (1997), 1462-1468;Cole-Strauss et al., Science 273, (1996), 1386-1389; Beetham et al.,1999, PNAS 96, 8774-8778).

A part of the DNA components of the RNA-DNA oligonucleotide ishomologous to a nucleic acid sequence of an endogenous Class 3 branchingenzyme gene, but, in comparison with the nucleic acid sequence of aClass 3 branching enzyme gene, it has a mutation or contains aheterologous region, which is surrounded by the homologous regions.

By base pairing of the homologous regions of the RNA-DNA oligonucleotideand the endogenous nucleic acid molecule followed by homologousrecombination, the mutation or heterologous region contained in the DNAcomponents of the RNA-DNA oligonucleotide can be transferred into thegenome of a plant cell. This leads to the reduction of the activity ofone or more Class 3 branching enzymes.

The person skilled in the art knows that he can achieve the activity ofClass 3 branching enzymes by the expression of non-functionalderivatives, in particular transdominant mutants, of such proteins,and/or by the expression of antagonists/inhibitors of such proteins.

Antagonist/inhibitors of such proteins include, for example, antibodies,antibody fragments or molecules with similar bonding characteristics.For example, a cytoplasmatic scFv antibody has been used to modulate theactivity of the phytochrome A protein in genetically modified tobaccoplants (Owen, Bio/Technology 10 (1992), 790-4; Review: Franken, E,Teuschel, U. and Hain, R., Current Opinion in Biotechnology 8, (1997),411416; Whitelam, Trends Plant Sci. 1 (1996), 268-272; Conrad andManteufel, Trends in Plant Science 6, (2001), 399-402; De Jaeger et al.,Plant Molecular Biology 43, (2000), 419-428). The reduction of theactivity of a branching enzyme in potato plants by expressing a specificantibody has been described by Jobling et al. (Nature Biotechnology 21,(2003), 77-80). Here, the antibody was provided with a plastidiarytarget sequence so that the inhibition of proteins localised in plastidswas guaranteed.

In conjunction with the present invention, plant cells and plantsaccording to the invention can also be manufactured by the use ofso-called insertion mutagenesis (overview article: Thorneycroft et al.,2001, Journal of experimental Botany 52 (361), 1593-1601). Insertionmutagenesis is to be understood to mean particularly the insertion oftransposons or so-called transfer DNA (T-DNA) into a gene coding for aClass 3 branching enzyme, whereby, as a result of which, the activity ofa Class 3 branching enzyme in the cell concerned is reduced.

The transposons can be both those that occur naturally in the cell(endogenous transposons) and also those that do not occur naturally insaid cell but are introduced into the cell (heterologous transposons) bymeans of genetic engineering methods, such as transformation of thecell, for example. Changing the expression of genes by means oftransposons is known to the person skilled in the art. An overview ofthe use of endogenous and heterologous transposons as tools in plantbiotechnology is presented in Ramachandran and Sundaresan (2001, PlantPhysiology and Biochemistry 39, 234-252). The possibility of identifyingmutants in which specific genes have been inactivated by transposoninsertion mutagenesis is presented in an overview by Maes et al. (1999,Trends in Plant Science 4 (3), 90-96). The production of rice mutantswith the help of endogenous transposons is described by Hirochika (2001,Current Opinion in Plant Biology 4, 118-122). The identification ofmaize genes with the help of endogenous retrotransposons is presented,for example, by Hanley et al. (2000, The Plant Journal 22 (4), 557-566).The possibility of manufacturing mutants with the help ofretrotransposons and methods of identifying mutants are described byKumar and Hirochika (2001, Trends in Plant Science 6 (3), 127-134). Theactivity of technological transposons in different species has beendescribed both for dicotyledonous and for monocotyledonous plants: e.g.for rice (Greco et al., 2001, Plant Physiology 125, 1175-1177; Liu etal., 1999, Molecular and General Genetics 262, 413-420; Hiroyuki et al.,1999, The Plant Journal 19 (5), 605-613; Jeon und Gynheung, 2001, PantScience 161, 211-219), barley (2000, Koprek et al., The Plant Journal 24(2), 253-263) Arabidopsis thaliana (Aarts et al., 1993, Nature 363,715-717, Schmidt und Willmitzer, 1989, Molecular and General Genetics220, 17-24; Altmann et al., 1992, Theoretical and Applied Gentics 84,371-383; Tissier et al., 1999, The Plant Cell 11, 1841-1852), tomato(Belzile und Yoder, 1992, The Plant Journal 2 (2), 173-179) and potato(Frey et al., 1989, Molecular and General Genetics 217, 172-177; Knappet al., 1988, Molecular and General Genetics 213, 285-290).

Basically, the plant cells according to the invention and the plantsaccording to the invention can be manufactured both with the help ofhomologous and heterologous transposons, whereby the use of homologoustransposons is also to be understood to mean those, which are naturallypresent in the corresponding wild type plant genome.

T-DNA insertion mutagenesis is based on the fact that certain sections(T-DNA) of Ti plasmids from Agrobacterium can integrate into the genomeof vegetable cells. The place of integration in the vegetable chromosomeis not defined, but can take place at any point. If the T-DNA integratesinto a part of the chromosome, which constitutes a gene function, thenthis can lead to a change in the gene expression and thus also to achange in the activity of a protein coded by the gene concerned. Inparticular, the integration of a T-DNA into the coded area of a proteinoften leads to the corresponding protein no longer being able to besynthesised at all, or no longer synthesised in active form, by the cellconcerned. The use of T-DNA insertions for producing mutants isdescribed, for example, for Arabidopsis thaliana (Krysan et al., 1999,The Plant Cell 11, 2283-2290; Atipiroz-Leehan and Feldmann, 1997, Trendsin genetics 13 (4), 152-156; Parinov and Sundaresan, 2000, CurrentOpinion in Biotechnology 11, 157-161) and rice (Jeon and An, 2001, PlantScience 161, 211-219; Jeon et al., 2000, The Plant Journal 22 (6),561-570). Methods for identifying mutants, which have been produced withthe help of T-DNA insertion mutagenesis, are described, amongst others,by Young et al., (2001, Plant Physiology 125, 513-518), Parinov et al.(1999, The Plant cell 11, 2263-2270), Thorneycroft et al. (2001, Journalof Experimental Botany 52, 1593-1601), and McKinney et al. (1995, ThePlant Journal 8 (4), 613-622).

T-DNA mutagenesis is basically suitable for the production of the plantcells and plants according to the invention, which have a reducedactivity of a Class 3 branching enzyme.

Surprisingly, it has been found that plant cells according to theinvention and plants according to the invention synthesise a modifiedstarch in comparison with starch of corresponding wild type plant cellsor wild type plants that have not been genetically modified.

The plant cells according to the invention and plants according to theinvention synthesise a modified starch, which in its physical-chemicalcharacteristics, in particular the amylose content or theamylose/amylopectin ratio, the degree of branching, the average chainlength, the side chain distribution, the viscosity behaviour, thegelling strength, the starch grain size and/or the starch grainmorphology, is changed in comparison with the synthesised starch in wildtype plant cells or plants, so that this is better suited for specialapplications.

It was surprisingly found that plant cells or plants of the inventionsynthesize a modified starch having decreased phosphate content. So farknown plants with a reduced activity of a branching enzyme (Class 1and/or Class 2) did show an increased phosphate content.

The present invention therefore also includes plant cells according tothe invention and plants according to the invention, which synthesise amodified starch.

In a preferred embodiment of the invention, the plant cells according tothe invention or the plant according to the invention synthesize astarch with a decreased phosphate content in comparison withcorresponding starch isolated from wild type plant cells or wild typeplants that have not been genetically modified. Preferably the plantcells according to the invention or the plants according to theinvention synthesize a starch having a total phosphate content that isdecreased by at least 10%, more preferably by at least 15% andparticular preferably by at least 20% in comparison with starch isolatedfrom corresponding wild type plant cells or wild type plants that havenot been genetically modified. Especially preferably the total phosphatecontent of starch isolated from plant cells of the invention or plantsof the invention is decreased by 14% to 22% in comparison with starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified.

In respect with C-6-phoaphate content the plant cells according to theinvention or the plants according to the invention synthesize a starchhaving a C-6-phosphate content that is decreased by at least 15%, morepreferably by at least 19% and particular preferably by at least 25% incomparison with starch isolated from corresponding wild type plant cellsor wild type plants that have not been genetically modified. Especiallypreferably the C-6-phoaphate content of starch isolated from plant cellsof the invention or plants of the invention is decreased by 15%% to 27%in comparison with starch isolated from corresponding wild type plantcells or wild type plants that have not been genetically modified.

Methods for the determination of total phosphate or C-6-phosphatecontent in starches are well known by a person skilled in the art.Preferred methods for the determination of total or C-6-phosphatecontent in starches to be used in combination with the present inventionare described below in the section general methods” (Starch analysis, e)Analysis of the side-chain distribution of the amylopectin by means ofion-exchange chromatography).

In a further prefered embodiment embodiment of the invention, the plantcells according to the invention or the plants according to theinvention synthesize a starch which has has an altered viscositybehaviour in comparison with starch isolated from corresponding wildtype plant cells or wild type plants that have not been geneticallymodified. Plant cells of the invention or plants of the inventionsynthesize a starch which has a decreased maximum viscosity, a decreasedminimum viscosity and/or a decreased final viscosity in comparison withstarch isolated from corresponding wild type plant cells or wild typeplants that have not been genetically modified.

The maximum viscosity of starch-isolated from plant cells of theinvention or plants of the invention is preferably decreased by at least8% and more preferably by at least 16% in comparison with starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified. Especially preferably themaximum viscosity of starch isolated from plant cells of the inventionor plants of the invention is decreased by 8% to 16% in comparison withstarch isolated from corresponding wild type plant cells or wild typeplants that have not been genetically modified.

The minimum viscosity of starch isolated from plant cells of theinvention or plants of the invention is preferably decreased by at least10%, more preferably by at least 15% and particularly preferably by atlest 25% in comparison with starch isolated from corresponding wild typeplant cells or wild type plants that have not been genetically modified.Especially preferably the minimum viscosity of starch isolated fromplant cells of the invention or plants of the invention is decreased by15% to 25% in comparison with starch isolated from corresponding wildtype plant cells or wild type plants that have not been geneticallymodified.

The final viscosity of starch isolated from plant cells of the inventionor plants of the invention is preferably decreased by at least 5% andmore preferably by at least 10% in comparison with starch isolated fromcorresponding wild type plant cells or wild type plants that have notbeen genetically modified. Particularly preferably the minimum viscosityof starch isolated from plant cells of the invention or plants of theinvention is decreased by 5% to 10% in comparison with starch isolatedfrom corresponding wild type plant cells or wild type plants that havenot been genetically modified.

It has further been found, that starch isolated from plant cells of theinvention or plants of the invention shows an increased gelling strengthin comparison with starch isolated from corresponding wild type plantcells or wild type plants that have not been genetically modified.

The present invention therefore also comprises plant cells of theinvention or plants of the invention that synthesize a starch with anincreased gel strength in comparison with starch isolated fromcorresponding wild type plant cells or wild type plants that have notbeen genetically modified. Preferably plant cells of the invention orplants of the invention synthesize a starch which shows a gel strengthwhich is increased by at least 20%, more preferably by at least 30% andparticular preferably by at least 35% in comparison with starch isolatedfrom corresponding wild type plant cells or wild type plants that havenot been genetically modified. Especially preferably the gel strength ofstarch isolated from plant cells of the invention or plants of theinvention is increased by 27% to 38% in comparison with starch isolatedfrom corresponding wild type plant cells or wild type plants that havenot been genetically modified.

Methods for the determination of viscosity behaviour or gellingproperties of starches are well known by a person skilled in the art.Preferred methods for the determination of viscosity behaviour orgelling properties of starches to be used in combination with thepresent invention are described below in the section “general methods”.

Furthermore it was surprisingly found that starch, isolated from plantcells of the invention or plants of the invention shows an altered sidechain distribution pattern in the amylopectin fraction in comparisonwith the amylopectin fraction from starch isolated from correspondingwild type plant cells or wild type plants that have not been geneticallymodified.

In a further embodiment of the invention, plant cells according to theinvention or the plants according to the invention synthesize a starchwith an altered short-side-chain distribution pattern in the amylopectinfraction in comparison with the amylopectin fraction from starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified. Preferably plant cellsaccording to the invention or the plants according to the inventionsynthesize a starch wherein the short-side-chains in the amylopectinfraction having a degree of polymerization (DP) of 6 and/or a DP of 7 isincreased in comparison with the amylopectin fraction from starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified. More preferably the amylopectinfraction of starch isolated form plant cells according to the inventionor plants according to the invention synthesize a starch whereinshort-side-chains with a DP 6 is increased by at least 15%, particularlypreferably by at least 20%, especially particularly by at least 25%and/or the short-side-chains with a DP 7 are increased by at least 2%,particularly preferably by at least 4%, especially preferably by atleast 8% in comparison with the amylopectin fraction from starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified.

In a further preferred embodiment of the invention the plant cellsaccording to the invention or the plants according to the inventionsynthesize a starch wherein the short-side-chains of DP 6 in theamylopectin fraction is increased by 17% to 29% and/or the side chainsof DP 7 in the amylopectin fraction is increased by 2% to 9% incomparison with the amylopectin fraction from starch isolated fromcorresponding wild type plant cells or wild type plants that have notbeen genetically modified.

In conjunction with the present invention, the term “short-side-chain”shall mean alpha-1,6-linked side-chains in the starch molecule having adegree of polymerization between DP 6 and DP 34.

Methods for the quantification of short-side-chains having a specifiedDP in the amylopectin fraction are well known by the person skilled inthe art. Preferred methods for the quantification of side-chains havinga specified DP, suitable to be used in combination with the presentinvention are described below in the section “general methods (Analysisof the side-chain distribution of the amylopectin by means ofion-exchange chromatography).

Furthermore it was found that the amylopectin fraction of starch,isolated from the plant cells according to the invention or the plantsaccording to the invention shows an altered total-side-chaindistribution.

Therefore, further embodiments of the present invention are the plantcells according to the invention or the plants according to theinvention which synthesize a starch wherein the groups oftotal-side-chains in the amylopectin fraction characterized by thefollowing ranges:

-   -   a) DP up to 11,    -   b) DP 12 to DP 19,    -   c) DP 20 to Dp 25 and/or    -   d) DP 26 to DP 31        is/are increased and/or the groups of total-side-chains in the        amylopectin fraction characterized by the following ranges:    -   a) DP 38 to DP 43    -   b) DP 44 to DP 49    -   c) DP 50 to DP 56    -   d) DP 57 to DP 62 and/or    -   e) DP 63 to DP 123        is/are decreased in comparison with the amylopectin fraction        from starch isolated from corresponding wild type plant cells or        wild type plants that have not been genetically modified.

In conjunction with the present invention, the term “total-side-chains”shall mean alpha-1,6-linked side-chains in the starch molecule having adegree of polymerization up to DP 123. A group of total-side-chainsconsists of all side-chains spanning a defined DP range (e.g. DP up to11, DP 12 to DP 19, DP 20 to Dp 25, DP 26 to DP 31, DP 38 to DP 43, DP44 to DP 49, DP 50 to DP 56, DP 57 to DP 62, DP 63 to DP 123).

Methods for the quantification of groups of total-side-chains spanningranges of side-chains with a specified DP in the amylopectin fractionare well known by the person skilled in the art. Preferred methods forthe quantification groups of total-side-chains, suitable to be used incombination with the present invention are described below in example5d).

Further embodiments of the invention are the plant cells according tothe invention or the plants according to the invention which synthesizea starch having a decreased peak onset Temperature (T₀), a decreasedpeak temperature (T Peak) and an increased delta H (dH) when analyzed bydifferential scanning calorimetrie (DSC) in comparison to starchisolated from corresponding wild type plant cells or wild type plantsthat have not been genetically modified.

Methods for the analysis of starch by DSC are well known by a personskilled in the art. Prefered Methods for DSC analysis suitable to beused in combination with the present invention are described below inthe section “general methods” (DSC-analysis (“Differential ScanningCalorimetry”).

Furthermore, genetically modified plants, which contain the plant cellsaccording to the invention, are also the subject matter of theinvention. Plants of this type can be produced from plant cellsaccording to the invention by regeneration.

In principle, the plants according to the invention can be plants of anyplant species, i.e. both monocotyledonous and dicotyledonous plants.Preferably they are useful plants, i.e. plants, which are cultivated bypeople for the purposes of food or for technical, in particularindustrial purposes.

In a further preferred embodiment, the plant according to the inventionis a starch-storing plant.

In a further preferred embodiment, the present invention relates tostarch-storing plants according to the invention of the genus Solanum,in particular Solanum tuberosum.

The term “starch-storing plants” includes all plants with starch-storingplant parts such as, for example, maize, rice, wheat, rye, oat, barley,cassaya, potato, sago, mung bean, pea or sorghum. Preferredstarch-storing plant parts are, for example, tubers, storage roots andgrains containing an endosperm; tubers are particularly preferred.

In conjunction with the present invention, the term “potato plant” or“potato” means plant species of the genus Solanum, in particulartuber-producing species of the genus Solanum and especially Solanumtuberosum.

The present invention also relates to propagation material of plantsaccording to the invention containing a plant cell according to theinvention.

Here, the term “propagation material” include those constituents of theplant that are suitable for producing offspring by vegetative or sexualmeans. Cuttings, callus cultures, rhizomes or tubers, for example, aresuitable for vegetative propagation. Other propagation materialincludes, for example, fruits, seeds, seedlings, protoplasts, cellcultures, etc. Preferably, the propagation material is seeds andparticularly preferably tubers.

In a further embodiment, the present invention relates to harvestableplant parts of plants according to the invention such as fruits, storageroots, roots, blooms, buds, shoots or stems, preferably seeds or tubers,wherein these harvestable parts contain at least one plant cellaccording to the invention.

Furthermore, the present invention also relates to a method for themanufacture of a plant according to the invention, wherein

-   -   a) a plant cell is genetically modified, whereby the genetic        modification leads to the reduction of the activity of a Class 3        vegetable branching enzyme in comparison with corresponding wild        type plant cells that have not been genetically modified;    -   b) a plant is regenerated from plant cells from Step a); and    -   c) if necessary, further plants are produced with the help of        the plants according to Step b).

The genetic modification introduced into the plant cell according toStep a) can basically be any type of genetic modification, which leadsto the reduction of the activity of a Class 3 branching enzyme.

The regeneration of the plants according to Step (b) can be carried outusing methods known to the person skilled in the art (e.g. described in“Plant Cell Culture Protocols”, 1999, edt. by R. D. Hall, Humana Press,ISBN 0-89603-549-2).

The production of further plants according to Step (c) of the methodaccording to the invention can be carried out, for example, byvegetative propagation (for example using cuttings, tubers or by meansof callus culture and regeneration of whole plants) or by sexualpropagation. Here, sexual propagation preferably takes place undercontrolled conditions, i.e. selected plants with particularcharacteristics are crossed and propagated with one another.

In a preferred embodiment of the method according to the invention, thegenetic modification consists in the introduction of a foreign nucleicacid molecule into the genome of the plant cell, wherein the presence orthe expression of said foreign nucleic acid molecule leads to a reducedactivity of a Class 3 branching enzyme in the cell.

The statements made in conjunction with plant cells according to theinvention and plants according to the invention apply with regard to the“introduction of a foreign nucleic acid molecule”.

In a further preferred embodiment, the method according to the inventionis used for producing potato plants according to the invention.

In a further preferred embodiment of the method according to theinvention, the foreign nucleic acid molecule is chosen from the groupconsisting of

-   -   a) Nucleic acid molecules, which code a protein with the amino        acid sequence given under Seq ID NO 4;    -   b) Nucleic acid molecules, which code a protein, the amino acid        sequence of which has an identity of at least 50% with the amino        acid sequence given under SEQ ID NO: 4;    -   c) Nucleic acid molecules, which include the nucleotide sequence        shown under Seq ID NO. 3 or a complimentary sequence;    -   d) Nucleic acid molecules, the nucleic acid sequence of which        has an identity of at least 50% with the nucleic acid sequences        described under a) or c);    -   e) Nucleic acid molecules, which hybridise with at least one        strand of the nucleic acid molecules described under a) or c)        under stringent conditions;    -   f) Nucleic acid molecules, the nucleotide sequence of which        deviates from the sequence of the nucleic acid molecules        identified under a), b), c), d), e) or f) due to the        degeneration of the genetic code; and    -   g) Nucleic acid molecules, which represent fragments, allelic        variants and/or derivatives of the nucleic acid molecules        identified under a), b), c), d), e) or f).

In a further preferred embodiment of the method according to theinvention, the foreign nucleic acid molecule is chosen from the groupconsisting of

-   -   a) Nucleic acid molecules, which code at least one antisense        RNA, which effects a reduction in the expression of at least one        endogenous gene, which codes a Class 3 branching enzyme;    -   b) Nucleic acid molecules, which by means of a co-suppression        effect lead to the reduction in the expression of at least one        endogenous gene, which codes a Class 3 branching enzyme;    -   c) Nucleic acid molecules, which code at least one ribozyme,        which splits specific transcripts of at least one endogenous        gene, which codes a Class 3 branching enzyme;    -   d) Nucleic acid molecules, which simultaneously code at least        one antisense RNA and at least one sense RNA, wherein the said        antisense RNA and the said sense RNA form a double-stranded RNA        molecule, which effects a reduction in the expression of at        least one endogenous gene, which codes a Class 3 branching        enzyme (RNAi technology);    -   e) Nucleic acid molecules introduced by means of in vivo        mutagenesis, which lead to a mutation or an insertion of a        heterologous sequence in at least one endogenous gene coding a        Class 3 branching enzyme, wherein the mutation or insertion        effects a reduction in the expression of a gene coding a Class 3        branching enzyme or results in the synthesis of inactive Class 3        branching enzymes;    -   f) Nucleic acid molecules, which code an antibody, wherein the        antibody results in a reduction in the activity of a Class 3        branching enzyme due to the bonding to a Class 3 branching        enzyme.    -   g) Nucleic acid molecules, which contain transposons, wherein        the integration of these transposons leads to a mutation or an        insertion in at least one endogenous gene coding a Class 3        branching enzyme, which effects a reduction in the expression of        at least one gene coding a Class 3 branching enzyme, or results        in the synthesis of inactive Class 3 branching enzymes; and/or    -   h) T-DNA molecules, which, due to insertion in at least one        endogenous gene coding a Class 3 branching enzyme, effect a        reduction in the expression of at least one gene coding a Class        3 branching enzyme, or result in the synthesis of inactive Class        3 branching enzyme.

In a further embodiment of the method according to the invention, thegenetically modified plants according to the invention synthesise amodified starch in comparison with corresponding wild type plants thathave not been genetically modified.

In a further embodiment of the method according to the invention, themethod according to the invention is used to manufacture plantsaccording to the invention.

The present invention also relates to the plants obtainable by themethod according to the invention.

It is also an object of the present invention to provide means such asDNA molecules, for example, for the production of plant cells accordingto the invention and plants according to the invention, which synthesisea modified starch in comparison with modified wild type plant cells orwild type plants that have not been genetically modified.

The present invention therefore also relates to nucleic acid moleculescoding for a protein with the enzymatic activity of a Class 3 branchingenzyme, chosen from the group consisting of

-   -   a) Nucleic acid molecules, which code a protein with the amino        acid sequence given under Seq ID NO 4;    -   b) Nucleic acid molecules, which code a protein, which includes        the amino acid sequence, which is coded by the insertion in        plasmid DSM 15926;    -   c) Nucleic acid molecules, which code a protein, the sequence of        which has an identity of at least 70% with the amino acid        sequence given under SEQ ID NO 4;    -   d) Nucleic acid molecules, which code a protein, the sequence of        which has an identity of at least 70% with the amino acid        sequence, which is coded by the insertion in plasmid DSM 15926;    -   e) Nucleic acid molecules, which include the nucleotide sequence        shown under Seq ID NO 3 or a complimentary sequence;    -   f) Nucleic acid molecules, which include the nucleotide sequence        of the insertion contained in plasmid DSM 15926;    -   g) Nucleic acid molecules, which have an identity of at least        70% with the nucleic acid sequences described under a), b), d)        or e);    -   h) Nucleic acid molecules, which hybridise with at least one        strand of the nucleic acid molecules described under a), b),        d), e) or f) under stringent conditions;    -   i) Nucleic acid molecules, the nucleotide sequence of which        deviates from the sequence of the nucleic acid molecules        identified under a), b), e) or f) due to the degeneration of the        genetic code; and    -   j) Nucleic acid molecules, which represent fragments, allelic        variants and/or derivatives of the nucleic acid molecules        identified under a), b), c), d), e), f), g), h) or i).

The amino acid sequence shown in SEQ ID NO 4 codes a protein with theactivity of a Class 3 branching enzyme from Solanum tuberosum.

The proteins coded from the different varieties of nucleic acidmolecules according to the invention have certain commoncharacteristics. These can include, for example, biological activity,molecular weight, immunological reactivity, conformation etc, as well asphysical characteristics such as, for example, the running behaviour ingel electrophoresis, chromatographic behaviour, sedimentationcoefficients, solubility, spectroscopic characteristics, stability;optimum pH, optimum temperature etc. The molecular weight of the Class 3branching enzyme from Solanum tuberosum derived from the amino acidsequence shown under SEQ ID NO 4 is ca. 103 kDa. The derived molecularweight of a protein according to the invention therefore preferably liesin the range from 85 kDa to 120 kDa, preferably in the range from 95 kDato 110 kDa and particularly preferably from ca. kDa 100 to 105 kDa.

The present invention relates to nucleic acid molecules, which code aprotein with the enzymatic activity of a Class 3 branching enzyme,wherein the coded protein has an identity of at least 70%, preferably ofat least 80%, particularly preferably of at least 90% and especiallypreferably of 95% with the amino acid sequence specified under SEQ ID NO4.

A plasmid containing a cDNA, which codes a Class 3 branching enzyme fromSolanum tuberosum, was deposited with the Deutsche Sammlung vonMikroorganismen und Zelikulturen GmbH, Mascheroder Weg 1b, 38124Braunschweig, Germany, in accordance with the Budapest Treaty on 15thSep. 2003 under the number DSM 15926. The amino acid sequence shown SEQID NO 4 can be derived from the coding region of the cDNA sequenceintegrated in plasmid DSM 15926 and codes for a Class 3 branching enzymefrom Solanum tuberosum. The present invention therefore also relates tonucleic acid molecules, which code a protein with the enzymatic activityof a Class 3 branching enzyme, which includes the amino acid sequence,which is coded by the insertion in plasmid DSM 15926, wherein the codedprotein has an identity of at least 70%, preferably of at least 80%,particularly preferably of at least 90% and especially preferably of 95%with the amino acid sequence, which can be derived from the insertion inDSM 15926.

The nucleic acid sequence shown SEQ ID NO 3 is a cDNA sequence, whichincludes the coding region for a Class 3 branching enzyme from Solanumtuberosum.

The present invention therefore also relates to nucleic acid molecules,which code a Class 3 branching enzyme and the coding region of thenucleotide sequence shown under Seq ID NO 3 or a complimentary sequence,nucleic acid molecules, which include the coding region of thenucleotide sequence of the insertion contained in plasmid DSM 15926 andnucleic acid molecules, which have an identity of at least 70%,preferably of at least 80%, particularly preferably of at least 90% andespecially preferably of at least 95% with the said nucleic acidmolecules.

With the help of the sequence information of the nucleic acid moleculeaccording to the invention or with the help of the nucleic acid moleculeaccording to the invention, it is now possible for the person skilled inthe art to isolate homologous sequences from other plant species,preferably from starch-storing plants, preferably from plant species ofthe genus Solanum, particularly preferably from Solanum tuberosum. Thiscan be carried out, for example, with the help of conventional methodssuch as the examination of cDNA or genomic banks with suitablehybridisation samples. The person skilled in the art knows thathomologous sequences can also be isolated with the help of (degenerated)oligonucleotides and the use of PCR-based methods.

The examination of databases, such as are made available, for example,by EMBL (http://www.ebi.ac.uk/Tools/index.htm) or NCBI (National Centerfor Biotechnology Information, http://www.ncbi.nim.nih.gov/), can alsobe used for identifying homologous sequences, which code for a Class 3branching enzyme. In this case, one or more sequences are specified as aso-called query. This query sequence is then compared by means ofstatistical computer programs with sequences, which are contained in theselected databases. Such database queries (e.g. blast or fasta searches)are known to the person skilled in the art and can be carried out byvarious providers.

If such a database query is carried out, e.g. at the NCBI (NationalCenter for Biotechnology Information, http://www.ncbi.nim.nih.gov/),then the standard settings, which are specified for the particularcomparison inquiry, should be used. For protein sequence comparisons(blastp), these are the following settings: Limit entrez=not activated;Filter=low complexity activated; Expect value=10; word size=3;Matrix=BLOSUM62; Gap costs: Existence=11, Extension=1.

For nucleic acid sequence comparisons (blastn), the following parametersmust be set: Limit entrez=not activated; Filter=low complexityactivated; Expect value=10; word size=11.

With such a database search, the sequences described in the presentinvention can be used as a query sequence in order to identify furthernucleic acid molecules and/or proteins, which code a Class 3 branchingenzyme.

With the help of the described methods, it is also possible to identifyand/or isolate nucleic acid molecules according to the invention, whichhybridise with the sequence specified under SEQ ID NO 3 and which code aClass 3 branching enzyme.

Within the framework of the present invention, the term “hybridising”means hybridisation under conventional hybridisation conditions,preferably under stringent conditions such as, for example, aredescribed in Sambrock et al., Molecular Cloning, A Laboratory Manual,2nd Ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.). Particularly preferably, “hybridising” means hybridisation underthe following conditions:

Hybridisation Buffer:

2×SSC; 10× Denhardt solution (Ficoll 400+PEG+BSA; Ratio 1:1:1); 0.1%SDS; 5 mM EDTA; 50 mM Na₂HPO₄; 250 μg/ml herring sperm DNA; 50 μg/mltRNA; or 25 M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS

Hybridisation temperature: T=65 to 68° C.

Wash buffer: 0.2×SSC; 0.1% SDS

Wash temperature: T=65 to 68° C.

In principle, nucleic acid molecules, which hybridise with the nucleicacid molecules according to the invention, can originate from any plantspecies, which expresses an appropriate protein, preferably theyoriginate from starch-storing plants, preferably from species of thegenus Solanum, particularly preferably from Solanum tuberosum. Nucleicacid molecules, which hybridise with the molecules according to theinvention, can, for example, be isolated from genomic or from cDNAlibraries. The identification and isolation of nuclear acid molecules ofthis type can be carried out using the nucleic acid molecules accordingto the invention or parts of these molecules or the reverse complementsof these molecules, e.g. by means of hybridisation according to standardmethods (see, for example, Sambrook et al., 1989, Molecular Cloning, ALaboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.) or by amplification using PCR.

Nucleic acid molecules, which exactly or essentially have the nucleotidesequence specified under SEQ ID NO 3 or parts of this sequence, can beused as hybridisation samples. The fragments used as hybridisationsamples can also be synthetic fragments or oligonucleotides, which havebeen manufactured using established synthesising techniques and thesequence of which corresponds essentially with that of a nucleic acidmolecule according to the invention. If genes have been identified andisolated, which hybridise with the nucleic acid sequences according tothe invention, then a determination of this sequence and an analysis ofthe characteristics of the proteins coded by this sequence should becarried out in order to establish whether a Class 3 branching enzyme isinvolved. Homology comparisons on the level of the nucleic acid or aminoacid sequence and a determination of the enzymatic activity areparticularly suitable for this purpose. As described above, the activityof a Class 3 branching enzyme can take place by expression in E. colistrains, which themselves do not express an active branching enzyme(Kiel et al., 1987 Mol. Gen. Genet 207: 294-301); Guan et al., 1995,Proc. Natl. Acad. Sci. 92, 964-967).

The molecules hybridising with the nucleic acid molecules according tothe invention particularly include fragments, derivatives and allelicvariants of the nucleic acid molecules according to the invention, whichcode a Class 3 branching enzyme from plants, preferably fromstarch-storing plants, preferably from plant species of the genusSolanum, particularly preferably from Solanum tuberosum. In conjunctionwith the present invention, the term “derivative” means that thesequences of these molecules differ at one or more positions from thesequences of the nucleic acid molecules described above and have a highdegree of identity with these sequences. Here, the deviation from thenucleic acid molecules described above can have come about, for example,due to deletion, addition, substitution, insertion or recombination.

Furthermore, identity means that functional and/or structuralequivalence exists between the nucleic acid molecules concerned or theproteins coded by them. The nucleic acid molecules, which are homologousto the molecules described above and constitute derivatives of thesemolecules, are generally variations of these molecules, which constitutemodifications, which execute the same biological function. At the sametime, the variations can occur naturally, for example they can besequences from other plant species, or they can be mutations, whereinthese mutations may have occurred in a natural manner or have beenintroduced by objective mutagenesis. The variations can also besynthetically manufactured sequences. The allelic variants can be bothnaturally occurring variants and also synthetically manufacturedvariants or variants produced by recombinant DNA techniques. Nucleicacid molecules, which deviate from nucleic acid molecules according tothe invention due to degeneration of the genetic code, constitute aspecial form of derivatives.

The proteins coded from the different derivatives of nucleic acidmolecules according to the invention have certain commoncharacteristics. These can include, for example, biological activity,substrate specificity, molecular weight, immunological reactivity,conformation etc, as well as physical characteristics such as, forexample, the running behaviour in gel electrophoresis, chromatographicbehaviour, sedimentation coefficients, solubility, spectroscopiccharacteristics, stability; optimum pH, optimum temperature etc.

The nucleic acid molecules according to the invention can be any nucleicacid molecules, in particular DNA or RNA molecules, for example cDNA,genomic DNA, mRNA etc. They can be naturally occurring molecules ormolecules manufactured by genetic or chemical synthesis methods. Theycan be single-stranded molecules, which either contain the coding or thenon-coding strand, or double-stranded molecules.

Furthermore, the present invention relates to nucleic acid molecules ofat least 21, preferably more than 50 and particularly preferably morethan 200 nucleotides length, which specifically hybridise with at leastone nucleic acid molecule according to the invention. Here, specificallyhybridise means that these molecules hybridise with nucleic acidmolecules, which code a protein according to the invention, but not withnucleic acid molecules, which code other proteins. In particular, theinvention relates to such nucleic acid molecules, which hybridise withtranscripts of nucleic acid molecules according to the invention and, asa result, can hinder their translation. Such nucleic acid molecules,which specifically hybridise with the nucleic acid molecules accordingto the invention, can, for example, be constituents of antisense, RNAior cosuppression constructs or ribozymes, or can be used as primers forPCR amplification.

In conjunction with the present invention, the term “identity” means asequence identity over the whole length of the coding region of at least60%, in particular an identity of at least 70%, preferably greater than80%, particularly preferably greater than 90% and especially of at least95%. In conjunction with the present invention, the term “identity” isto be understood to mean the number of amino acids/nucleotides(identity) corresponding with other proteins/nucleic acids, expressed asa percentage. Identity is preferably determined by comparing the Seq. IDNO 4 or SEQ ID NO 3 with other proteins/nucleic acids with the help ofcomputer programs. If sequences that are compared with one another havedifferent lengths, the identity is to be determined in such a way thatthe number of amino acids, which have the shorter sequence in commonwith the longer sequence, determines the percentage quotient of theidentity. Preferably, identity is determined by means of the computerprogram ClustalW, which is well known and available to the public(Thompson et al., Nucleic Acids Research 22 (1994), 4673-4680). ClustalWis made publicly available by Julie Thompson(Thompson@EMBL-Heidelberg.DE) and Toby Gibson(Gibson@EMBL-Heidelberg.DE), European Molecular Biology Laboratory,Meyerhofstrasse 1, D 69117 Heidelberg, Germany. ClustalW can also bedownloaded from different Internet sites, including the IGBMC (Institutde Génétique et de Biologie Moléculaire et Cellulaire, B.P.163, 67404Illkirch Cedex, France; ftp://ftp-igbmc.u-strasbg.fr/pub/) and the EBI(ftp://ftp.ebi.ac.uk/pub/software/) as well as from all mirroredInternet sites of the EBI (European Bioinformatics Institute, WellcomeTrust Genome Campus, Hinxton, Cambridge CB10 1SD, UK).

Preferably, Version 1.8 of the ClustalW computer program is used todetermine the identity between proteins according to the invention andother proteins. In doing so, the following parameters must be set:KTUPLE=1, TOPDIAG=5, WINDOW=5, PAIRGAP=3, GAPOPEN=10, GAPEXTEND=0.05,GAPDIST=8, MAXDIV=40, MATRIX=GONNET, ENDGAPS(OFF), NOPGAP, NOHGAP.

Preferably, Version 1.8 of the ClustalW computer program is used todetermine the identity between the nucleotide sequence of the nucleicacid molecules according to the invention, for example, and thenucleotide sequence of other nucleic acid molecules. In doing so, thefollowing parameters must be set: KTUPLE=2, TOPDIAGS=4, PAIRGAP=5,DNAMATRIX:IUB, GAPOPEN=10, GAPEXT=5, MAXDIV=40, TRANSITIONS: unweighted.

Basically, nucleic acid molecules according to the invention, canoriginate from any plant, preferably they originate from starch-storingplants, preferably from plant species of the genus Solanum, particularlypreferably from Solanum tuberosum.

Furthermore, the invention relates to vectors, in particular plasmids,cosmids, viruses, bacteriophages and other common vectors in geneticengineering, which contain the nucleic acid molecules according to theinvention described above.

In a preferred embodiment, the nucleic acid molecules according to theinvention contained in the vectors are linked with regulatory sequences,which guarantee expression in prokaryontic or eukaryontic cells. Here,the term “expression” can mean both transcription as well astranscription and translation. In this case, the nucleic acid moleculesaccording to the invention can be present in “sense” orientation and/orin “antisense” orientation to the regulatory sequences.

Regulatory sequences for expression in prokaryontic organisms, e.g. E.coli, and in eukaryontic organisms are adequately described in theliterature, in particular those for expression in yeast such asSaccharomyces cerevisiae, for example. An overview of differentexpression systems for proteins in different host organisms can befound, for example, in Methods in Enzymology 153 (1987), 383-516 and inBitter et al. (Methods in Enzymology 153 (1987), 516-544).

For expressing the nucleic acid molecules, which code a Class 3branching enzyme, in sense and/or antisense orientation in vegetablecells, these are preferably linked with regulatory DNA sequences, whichguarantee transcription in vegetable cells. In particular, these includepromoters. In general, any promoter that is active in vegetable cells iseligible for expression.

At the same time, the promoter can be chosen so that expression takesplace constitutively or only in a certain tissue, at a certain stage ofthe plant development or at a time determined by external influences.The promoter can be homologous or heterologous both with respect to theplant and with respect to the nucleic acid molecule.

Suitable promoters are, for example, the promoter of the 35S RNA of thecauliflower mosaic virus and the ubiquitin promoter from maize forconstitutive expression, the patatin promoter 833 (Rocha-Sosa et al.,EMBO J. 8 (1989), 23-29) for tuber-specific expression in potatoes or apromoter, which only ensures expression in photosynthetically activetissues, e.g. the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad.Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO J. 8 (1989),2445-2451) or, for endosperm-specific expression of the HMG promoterfrom wheat, the USP promoter, the phaseolin promoter, promoters of zeingenes from maize (Pedersen et al., Cell 29 (1982), 1015-1026; Quatroccioet al., Plant Mol. Biol. 15 (1990), 81-93), glutelin promoter (Leisy etal., Plant Mol. Biol. 14 (1990), 41-50; Zheng et al., Plant J. 4 (1993),357-366; Yoshihara et al., FEBS Lett. 383 (1996), 213-218) or shrunken-1promoter (Werr et al., EMBO J. 4 (1985), 1373-1380). However, promoterscan also be used, which are only activated at a time determined byexternal influences (see for example WO 9307279). Promoters ofheat-shock proteins, which allow simple induction, can be of particularinterest here. Furthermore, seed-specific promoters can be used, such asthe USP promoter from Vicia faba, which guarantees seed-specificexpression in Vicia faba and other plants (Fiedler et al., Plant Mol.Biol. 22 (1993), 669-679; Bäumlein et al., Mol. Gen. Genet. 225 (1991),459-467).

Furthermore, a termination sequence (polyadenylation signal) can bepresent, which is used for adding a poly-A tail to the transcript. Afunction in the stabilisation of the transcripts is ascribed to thepoly-A tail. Elements of this type are described in the literature (cf.Gielen et al., EMBO J. 8 (1989), 23-29) and can be exchanged at will.

In a further embodiment, the present invention relates to vectors, whichcontain DNA molecules, which code at least one antisense RNA, whicheffects a reduction in the expression of at least one endogenous gene,which codes a Class 3 branching enzyme.

In a further special embodiment, the present invention relates tovectors, which contain DNA molecules, which by means of a cosuppressioneffect lead to a reduction in the expression of at least one endogenousgene, which codes a Class 3 branching enzyme.

In a further embodiment, the present invention relates to vectors, whichcontain DNA molecules, which code at least one ribozyme, which splitsspecific transcripts of at least one endogenous gene, which codes aClass 3 branching enzyme.

In a further embodiment, the present invention relates to vectors, whichcontain DNA molecules, which simultaneously code at least one antisenseRNA and at least one sense RNA, wherein the said antisense RNA and thesaid sense RNA form a double-stranded RNA molecule, which effects areduction in the expression of at least one endogenous gene, which codesa Class 3 branching enzyme (RNAi technology).

A further subject of the present invention is a host cell, in particulara prokaryontic or eukaryontic cell, which is genetically modified with anucleic acid molecule according to the invention and/or with a vectoraccording to the invention, as well as cells, which originate from hostcells of this type and which contain the genetic modification accordingto the invention.

In a preferred embodiment, the invention relates to host cells, inparticular prokaryontic or eukaryontic cells, which have beentransformed using the nucleic acid molecule according to the inventionor a vector according to the invention, as well as host cells, whichoriginate from host cells of this type and which contain the describednucleic acid molecules or vectors according to the invention.

The host cells can be bacteria (e.g. E. coli) or fungus cells (e.g.yeast, in particular S. cerevisiae, Agaricus, in particular Agaricusbisporus), as well as vegetable or animal cells. Here, the term“transforms” means that the cells according to the invention aregenetically modified with a nucleic acid molecule according to theinvention inasmuch as they contain at least one nucleic acid moleculeaccording to the invention in addition to their natural genome. This canbe freely present in the cell, possibly as a self-replicating molecule,or it can be stably integrated in the genome of the host cell. The hostcells are preferably microorganisms. Within the framework of the presentapplication, these are understood to mean all bacteria and all protista(e.g. fungi, in particular yeast and algae), as defined, for example, inSchlegel “Aligemeine Mikrobiologie” (Georg Thieme Verlag (1985), 1-2).

It is especially preferred if the host cells according to the inventionare plant cells. In principle, these can be plant cells from any plantspecies, i.e. both monocotyledonous and dicotyledonous plants.Preferably, these will be plant cells from useful agricultural plants,i.e. from plants, which are cultivated by people for the purposes offood or for technical, in particular industrial purposes. The inventionrelates preferably to plant cells and plants from starch-storing plants(maize, rice, wheat, rye, oat, barley, cassaya, potato, sago, mung bean,pea or sorghum); in particular, plant cells from maize, rice, wheat orpotato plants are particularly preferred.

A further subject of the present invention are proteins with theenzymatic activity of a Class 3 branching enzyme, chosen from the groupconsisting of

-   -   a) Proteins, which include the amino acid sequence specified        under SEQ ID NO 4;    -   b) Proteins, which are coded by the coding region of the DNA        inserted in the plasmid DSM 15926; or    -   c) Proteins, which have an identity of at least 70% with the        amino acid sequence of the proteins identified under a) or b).

In a further embodiment, the present invention relates to proteins withthe enzymatic activity of a Class 3 branching enzyme, wherein the codedprotein has an identity of at least 70%, preferably of at least 80%,particularly preferably of at least 90% and especially preferably of 95%with the amino acid sequence specified under SEQ ID NO 4 or with theamino acid sequence of a Class 3 branching enzyme coded by the insertionin plasmid DSM 15926.

In a further embodiment, the invention also relates to proteins, whichare coded by nucleic acid molecules according to the invention.

In a preferred embodiment, the present invention relates to a proteinwith the enzymatic activity of a Class 3 branching enzyme, wherein theClass 3 branching enzyme originates from a potato plant.

Surprisingly, it has been found that plant cells and plants, which havea reduced activity of a Class 3 branching enzyme, synthesise a starch,which is modified in comparison with starch from wild type plant cellsor wild type plants.

In conjunction with the present invention, the term “modified starch”means that the starch has changed physical-chemical characteristicscompared with non-modified starch obtainable from corresponding wildtype plant cells or wild type plants that have not been geneticallymodified.

In a preferred embodiment of the present invention, the modified starchis native starch.

In conjunction with the present invention, the term “native starch”means that the starch is isolated from plants according to theinvention, harvestable plant plants according to the invention orpropagation material of plants according to the invention by methodsknown to the person skilled in the art.

Starch is a classical additive for many foodstuffs in which itessentially takes over the function of binding aqueous additives orincreasing the viscosity, or brings about an increased formation of gel.Important characteristic features are the flow and sorption behaviour,the source and sticking temperature, the viscosity and thickeningperformance, the solubility of the starch, the transparency and pastestructure, the heat, shearing and acidic stability, the tendency toretrogradation, the ability to form a film, the freezing/thawingstability, the digestibility as well as the ability to form complexeswith, for example, inorganic or organic ions.

In the area of the non-foodstuffs industry, starch can be used, forexample, as an auxiliary substance for different manufacturing processesor as an additive in technical products. Particular mention must be madehere of the paper and cardboard industry where starch is used as anauxiliary substance. Here, the starch is primarily used for retardation(holding back of solids), the bonding of filler and fine materialparticles, as a consolidation material and for dehydration. In additionto this, the favourable characteristics of starch with regard tostiffness, hardness, sound, grip, shine, smoothness and resistance tosplitting as well as the surfaces are also fully utilised.

A further major area of use of starches is in the adhesive industry,where the possible applications are divided into four sub-areas. Use asa pure starch adhesive, use with starch adhesives prepared with specialchemicals, use of starch as an additive to synthetic resins and polymerdispersions, and the use of starches as a stretching medium forsynthetic adhesives.

Furthermore, starches can be used as additives for building materials(e.g. plasterboard sheets, ready-mixed concrete, plaster and mineralfibres), for the manufacture of media for stabilising soil, as afunctional aid in plant protection media or fertilisers, as a functionalaid in the pharmaceutical industry (e.g. as a bonding medium, tabletdispersal medium, in lubricating and vulnerary powders) and the cosmeticindustry (as a carrier of additives), as a strengthening additive forcoal and briquettes, as a flocculation medium (e.g. in the preparationof carbon sludge) and as a bonding medium, e.g. in Betonit.

Plant cells according to the invention and plants according to theinvention synthesise a modified starch in comparison with starch ofcorresponding wild type plant cells or wild type plants that have notbeen genetically modified. In its physical-chemical characteristics,e.g. the amylopectin/amylose ratio, the degree of branching, thephosphate content, the average chain length, the viscosity behaviour,the starch grain size, the side chain distribution and/or the starchgrain form, the modified starch is changed in comparison with thesynthesised starch in wild type plant cells or plants so that it isbetter suited for use in particular application areas, for example.

The present invention therefore also relates to modified starchesobtainable or isolated from plant cells according to the invention orplants according to the invention, from propagation material accordingto the invention or from harvestable plant parts according to theinvention.

In a particularly preferred embodiment, the present invention relates tomodified potato starch.

Furthermore the present invention relates to a method for themanufacture of a modified starch including the step of extracting thestarch from a plant cell according to the invention or from a plantaccording to the invention, from propagation material according to theinvention of such a plant and/or from harvestable plant parts accordingto the invention of such a plant, preferably from starch-storing partsaccording to the invention of a plant. Preferably, such a method alsoincludes the step of harvesting the cultivated plants or plant partsand/or the propagation material of these plants before the extraction ofthe starch and, further, particularly preferably the step of cultivatingplants according to the invention before harvesting.

Methods for extracting starches from plants or from starch-storing partsof plants are known to the person skilled in the art. Furthermore,methods for extracting starch from different starch-storing plants aredescribed, e.g. in Starch: Chemistry and Technology (Publisher:Whistler, BeMiller and Paschall (1994), 2nd Edition, Academic Press Inc.London Ltd; ISBN 0-12-746270-8; see e.g. Chapter XII, Page 412-468:Maize and Sorghum Starches: Manufacture; by Watson; Chapter XIII, Page469-479: Tapioca, Arrowroot and Sago Starches: Manufacture; byCorbishley and Miller; Chapter XIV, Page 479-490: Potato starch:Manufacture and Uses; by Mitch; Chapter XV, Page 491 to 506: Wheatstarch: Manufacture, Modification and Uses; by Knight and Oson; andChapter XVI, Page 507 to 528: Rice starch: Manufacture and Uses; byRohmer and Klem; Maize starch: Eckhoff et al., Cereal Chem. 73 (1996),54-57, the extraction of maize starch on an industrial scale isgenerally achieved by so-called “wet milling”.). Devices, which are incommon use in methods for extracting starch from plant material areseparators, decanters, hydrocyclones, spray dryers and fluid bed dryers.

In conjunction with the present invention, the term “starch-storingparts” is to be understood to mean such parts of a plant in which, incontrast to transitory leaf starch, starch is stored as a deposit forsurviving for longer periods. Preferred starch-storing parts are tubers,storage roots, seeds or endosperm; particularly preferred are potatotubers or the endosperm of maize, wheat or rice plants.

Modified starch obtainable by the method according to the invention isalso the subject matter of the present invention.

Furthermore, the use of plant cells according to the invention or plantsaccording to the invention for manufacturing a modified starch are thesubject matter of the present invention.

The person skilled in the art knows that the characteristics of starchcan be changed by thermal, chemical, enzymatic or mechanical derivation,for example. Derived starches are particularly suitable for differentapplications in the foodstuffs and/or non-foodstuffs sector. Thestarches according to the invention are better suited as a startingsubstance for the manufacture of derived starches than conventionalstarches. In the manufacture of derived starch, they are distinguishedby better processing capability and lead to new products, as a modifiedstarch is used as a new starting material for the derivation process.

The present invention therefore also relates to the manufacture of aderived starch, wherein modified starch according to the invention isderived retrospectively.

In conjunction with the present invention, the term “derived starch” isto be understood to mean a modified starch according to the invention,the characteristics of which have been retrospectively changed afterisolation from vegetable cells with the help of chemical, enzymatic,thermal or mechanical methods.

In a preferred embodiment of the present invention, the derived starchaccording to the invention is starch that has been heat-treated and/oracid-treated.

In a further preferred embodiment, the derived starches are starchethers, in particular starch alkyl ethers, O-allyl ethers, hydroxylalkylethers, O-carboxylmethyl ethers, nitrogen-containing starch ethers,phosphate-containing starch ethers or sulphur-containing starch ethers.

In a further preferred embodiment, the derived starches are cross-linkedstarches.

In a further preferred embodiment, the derived starches are starch graftpolymers.

In a further preferred embodiment, the derived starches are oxidisedstarches.

In a further preferred embodiment, the derived starches are starchesters, in articular starch esters, which have been introduced into thestarch using organic acids. Particularly preferably these are phosphate,nitrate, sulphate, xanthate, acetate or citrate starches.

The derived starches according to the invention are suitable fordifferent applications in the foodstuffs and/or non-foodstuffs sector.Methods for manufacturing derived starches according to the inventionare known to the person skilled in the art and are adequately describedin the general literature. An overview on the manufacture of derivedstarches can be found, for example, in Orthoefer (in Corn, Chemistry andTechnology, 1987, eds. Watson und Ramstad, Chapter 16, 479-499).

Derived starch obtainable by the method according to the invention formanufacturing a derived starch is also the subject matter of the presentinvention.

Furthermore, the use of modified starches according to the invention formanufacturing derived starch is the subject matter of the presentinvention.

Description of Sequences

SEQ ID NO 1: Nucleic acid sequence containing the coding region of the3′-area of a Class 3 branching enzyme from Solanum tuberosum (cvDésirée). This sequence is inserted in plasmid AN 46-196.

SEQ ID NO 2: Nucleic acid sequence containing the coding region of the5′-area of a Class 3 branching enzyme from Solanum tuberosum (cvDésirée). This sequence is inserted in plasmid AN 47-196.

SEQ ID NO 3: Nucleic acid sequence containing the full coding region ofa Class 3 branching enzyme from Solanum tuberosum (cv Désirée). Thissequence is inserted in plasmid AN 49 and was deposited with theDeutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, MascheroderWeg 1b, 38124 Braunschweig, Germany, in accordance with the BudapestTreaty on 15th Sep. 2003 under the number DSM 15926.

SEQ ID NO 4: Amino acid sequence coding a Class 3 branching enzyme fromSolanum tuberosum (cv Désirée). This sequence can be derived from thenucleic acid sequence inserted in plasmid AN 49 or from the nucleic acidsequence described under SEQ ID NO 3.

SEQ ID NO 5: Nucleic acid sequence containing the full coding region ofa Class 3 branching enzyme from Solanum tuberosum (cv Désirée). Thissequence has been obtained by combining the nucleic acid sequencesdescribed under SEQ ID NO 1 and SEQ ID NO 2. This nucleic acid sequenceconstitutes an allelic variant of the nucleic acid sequence describedunder SEQ ID NO 3 coding a Class 3 branching enzyme.

SEQ ID NO 6: Amino acid sequence coding a Class 3 branching enzyme fromSolanum tuberosum (cv Désirée). This sequence can be derived from thenucleic acid sequence described under SEQ ID NO 5 and constitutes anallelic variant of the amino acid sequence described under SEQ ID NO 4coding a Class 3 branching enzyme

General Methods

The following methods were used in the examples:

Demonstration of the Activity of a Class 3 Branching Enzyme

The activity of a Class 3 branching enzyme was demonstrated with thehelp of non-denaturing gel electrophoresis as follows:

To isolate proteins from plants, the test material was ground with apestle in liquid nitrogen, absorbed into an extraction buffer (50 mM Nacitrate, pH 6.5; 1 mM EDTA, 4 mM DTT) and, after centrifugation (10 min,14.000 g, 4° C.), was used directly for measurement of the proteincontent according to Bradford. Subsequently, 5 μg to 20 μg total proteinextract was mixed with 4× loading buffer (20% glycerol, 125 mM Tris HCl,pH 6.8) and loaded onto a BE activity gel. The BE activity gel was madeup as follows: 2.5 ml 30% acrylamide:0.8% bisacrylamide, 0.1 ml runningbuffer, 7.4 ml H₂O, 10% ammonium persulphate solution and 5 μlN,N,N′,N′-tetramethylethylenediamine (TEMED). The running buffer (RB)was made up as follows: RB=30.2 g Tris base, pH 8.0, 144 g glycine on 1L H₂O. On completion of the gel run, each of the gels was incubatedovernight at 37° C. in 25 ml “phosphorylase buffer” (25 ml 1M Na citratepH 7.0, 0.47 g glucose-1-phosphate, 12.5 mg AMP, 2.5 mg phosphorylasea/b from “rabbit”). The gels were coloured with Lugol's solution.

Starch Analysis

a) Determination of the Amylose Content and of the Amylose/AmylopectinRatio

Starch was isolated from potato plants by standard methods, and theamylose content and the amylose:amylopectin ratio was determined by themethod described by Hovenkamp-Hermelink et al. (Potato Research 31,(1988), 241-246).

b) Determination of the Phosphate Content

In starch, the positions C2, C3 and C6 of the glucose units can bephosphorylated. To determine the C6-P content of starch, 50 mg of starchare hydrolysed for 4 h at 95° C. in 500 μl of 0.7 M HCl. The samples arethen centrifuged for 10 minutes at 15500×g and the supernatants areremoved. 7 μl of the supernatants are mixed with 193 μl of imidazolebuffer (100 mM imidazole, pH 7.4; 5 mM MgCl₂, 1 mM EDTA and 0.4 mM NAD).The measurement was carried out in a photometer at 340 nm. After thebase absorption had been established, the enzyme reaction was started byaddition of 2 units glucose-6-phosphate dehydrogenase (from Leuconostocmesenteroides, Boehringer Mannheim). The change in absorption isdirectly proportional to the concentration of the G-6-P content of thestarch.

The total phosphate content was determined by the method of Ames(Methods in Enzymology VIII, (1966), 115-118).

Approximately −50 mg of starch are treated with 30 μl of ethanolicmagnesium nitrate solution and ashed for 3 hours at 500° C. in a muffleoven. The residue is treated with 300 μl of 0.5 M hydrochloric acid andincubated for 30 minutes at 60° C. One aliquot is subsequently made upto 300 μl 0.5 M hydrochloric acid and this is added to a mixture of 100μl of 10% ascorbic acid and 600 μl of 0.42% ammonium molybdate in 2 Msulphuric acid and incubated for 20 minutes at 45° C.

This is followed by a photometric determination at 820 nm with aphosphate calibration series as standard.

c) Determination of the Viscosity Characteristics by Means of a RapidVisco Analyser (RVA)

2 g of starch (DM) are taken up in 25 ml of H₂O (VE-type water,conductivity of at least 15 mega ohm) and used for the analysis in aRapid Visco Analyser Super3 (Newport Scientific Pty Ltd., InvestmetSupport Group, Warriewod NSW 2102, Australia). The apparatus is operatedfollowing the manufacturer's instructions. The viscosity values areindicated in Centipoise (cP) in accordance with the manufacturer'soperating manual, which is incorporated into the description herewith byreference. To determine the viscosity of the aqueous starch solution,the starch suspension is first stirred for 10 seconds at 960 rpm andsubsequently heated at 50° C. at a stirring speed of 160 rpm, initiallyfor a minute (step 1). The temperature was then raised from 50° C. to95° C. at a heating rate of 12° C. per minute (step 2). The temperatureis held for 2.5 minutes at 95° C. (step 3) and then cooled from 95° C.to 50° C. at 12° C. per minute (step 4). In the last step (step 5), thetemperature of 50° C. is held for 2 minutes. The viscosity is determinedduring the entire duration.

After the programme has ended, the stirrer is removed and the beakercovered. The gelatinized starch is now available for the textureanalysis after 24 hours incubation at room temperature.

The profile of the RVA analysis contains parameters which are shown forthe comparison of different measurements and substances. In the contextof the present invention, the following terms are to be understood asfollows:

1. Maximum Viscosity (RVA Max)

The maximum viscosity is understood as meaning the highest viscosityvalue, measured in cP, obtained in step 2 or 3 of the temperatureprofile.

2. Minimum Viscosity (RVA Min)

The minimum viscosity is understood as meaning the lowest viscosityvalue, measured in cP, observed in the temperature profile after themaximum viscosity. Normally, this takes place in step 3 of thetemperature profile.

3. Final Viscosity (RVA Fin)

The final viscosity is understood as meaning the viscosity value,measured in cP, observed at the end of the measurement.

4. Setback (RVA Set)

What is known as the “setback” is calculated by subtracting the value ofthe final viscosity from that of the minimum occurring after the maximumviscosity in the curve.

5. Gelatinization Temperature (RVA PT)

The gelatinization temperature is understood as meaning the point intime of the temperature profile where, for the first time, the viscosityincreases drastically for a brief period.

d) Determination of the Gel Strength (Texture Analyser)

2 g of starch (DM) are gelatinized in the RVA apparatus in 25 ml of anaqueous suspension (temperature programme: see item d) “Determination ofthe viscosity characteristics by means of a Rapid Visco Analyser (RVA)”)and subsequently stored for 24 hours at room temperature in a sealedcontainer. The samples are fixed under the probe (round piston withplanar surface) of a Texture Analyser TA-XT2 from Stable Micro Systems(Surrey, UK) and the gel strength was determined using the followingparameters: Test speed 0.5 mm/s Depth of penetration 7 mm Contactsurface 113 mm² Pressure 2 ge) Analysis of the Side-Chain Distribution of the Amylopectin by Meansof Ion-Exchange Chromatography

To separate amylose and amylopectin, 200 mg of starch are dissolved in50 ml reaction vessels, using 12 ml of 90% (v/v) DMSO in H₂0. Afteraddition of 3 volumes of ethanol, the precipitate is separated bycentrifugation for 10 minutes at about 100×g at room temperature (RT).The pellet is then washed with 30 ml of ethanol, dried and dissolved in40 ml of 1% (w/v) NaCl solution at 75° C. After the solution has cooledto 30° C., approximately 90 mg of thymol are added slowly, and thissolution is incubated for at least 60 h at 30° C. The solution is thencentrifuged for 30 minutes at 2000×g (RT). The supernatant is thentreated with 3 volumes of ethanol, and the amylopectin which settles outis separated by centrifugation for 5 minutes at 2000×g (RT). The pellet(amylopectin) is then washed with ethanol and dried using acetone. Byaddition of DMSO to the pellet, one obtains a 1% solution, of which 200μl are treated with 345 μl of water, 10 μl of 0.5 M sodium acetate (pH3.5) and 5 μl of isoamylase (dilution 1:10; Megazyme) and incubated forabout 16 hours at 37° C. A 1:5 aqueous dilution of this digest issubsequently filtered through a 0.2 μm filter, and 100 μl of thefiltrate are analysed by ion chromatography (HPAEC-PAD, Dionex).Separation was performed using a PA-100 column (with suitableprecolumn), while detection was performed amperometrically. The elutionconditions were as follows:

Solution A—0.15M NaOH

Solution B—1 M sodium acetate in 0.15M NaOH TABLE 1 Composition of theelution buffer for the side chain analysis of the amylopectin atdifferent times during the HPEAC-PAD Dionex analysis. Between the timesstated, the composition of the elution buffer changes in each caselinearly. t (min) Solution A (%) Solution B (%)  5 0 100 35 30 70 45 3268 60 100 0 70 100 0 72 0 100 80 0 100 Stop

The determination of the relative amount of short side chains in thetotal of all side chains is carried out via the determination of thepercentage of a particular side chain in the total of all side chains.The total of all side chains is determined via the determination of thetotal area under the peaks which represent the polymerization degrees ofDP 6 to 34 in the HPCL chromatogram.

The percentage of a particular side chain in the total of all sidechains is determined via the determination of the ratio of the areaunder the peak which represents this side chain in the HPLC chromatogramto the total area. The programme Chromelion 6.20 Version 6.20 fromDionex, USA, was used for determining the peak areas.

f) Determination of the Activity of the BEIII Protein

This was carried out as specified in the example.

g) DSC-Analysis (“Differential Scanning Calorimetry”)

Investigations with the aid of DSC-analysis have been done by the methoddescribed by WO 01/19975. 10 mg starch treated with 30 μl H₂O (VE-typewater, conductivity of at least 15 mega ohm) were sealed in stainlesssteal pans (volume 50 μl). The pan is heated from 20° C. to 150° C. at arate of 10° C. per minute in a Diamond DSC-instrument (Perkin Elmer).The programme Pyres from Perkin Elmer was used for determining the data.

EXAMPLES Example 1

Cloning of a Full-Length Sequence Coding a Class 3 Branching Enzyme fromSolanum tuberosum

The gene sequence coding for this Class 3 branching enzyme in Solanumtuberosum has not previously been described.

By sequence comparisons with different branching enzymes, a domain wasidentified, with the help of which EST databases were examined. In doingso, the EST TC73137 (TIGR database;http://www.tigr.org/tigr-scripts/tgi/tc_report.pl?tc=TC73137&species=potato)from potato was identified.

With the help of the primers B1_Asp (GAT GGG TAC CAG CAC TTC TAC TTG GCAGAG G) and B2_Sal (TCA AGT CGA CCA CM CCA GTC CAT TTC TGG), a sequencefrom a tuber-specific cDNA bank from Solanum tuberosum (cv. Désirée)corresponding to this EST sequence was amplified. Attempts to useleaf-specific, “sink”-tissue-specific or “source”-tissue-specific cDNAbanks as a template for the PCR reaction led to no amplification.

In order to amplify the whole coding sequence of the branching enzymeconcerned, which up to now had also included unknown sequences, primerswere manufactured, which were complimentary to the ends of thepreviously known sequence and vector sequences of the cDNA banksconcerned. With all the primer combinations for the amplification of afull-length sequence of a Class 3 branching enzyme used in thisapproach, it was not possible to amplify any further area. Hereupon, ESTdatabases of tomato were examined again.

In this case, two ESTs from tomato were identified (TIGR database;BG127920 and TC130382), which either had a high homology to theamplification of the Class 3 branching enzyme from potato describedabove (TC130382) and (BG127920) respectively, or to the putativebranching enzyme gene from arabidopsis (GenBank:GP|9294564|dbj|BAB02827.1).

Primers were now manufactured again in order to also amplify previouslyunknown sequences of the Class 3 branching enzyme. By means of PCR, the3′-area of the Class 3 branching enzyme was amplified from a cDNA bank,made from tubers of Solanum tuberosum (cv. Désirée), with the primersKM2_Spe (5′-TCAAACTAGTCACAACCAGTCCATTTCTGG-3′) and So_putE(5′-CACTTTAGAAGGTATCAGAGC-3′). The fragment with a size of ca. 1 kb thatwas obtained was cloned undirectedly in the pCR4-TOPO vector fromInvitrogen (product number: 45-0030). The plasmid produced wasdesignated as AN 46-196. The sequence of the inserted fragments in theplasmid AN 46-196 is shown under SEQ ID NO 1.

The 5′-area was likewise amplified by means of PCR technology and usingthe primers So_put5′ (5′-GTATTTCTGCGAAGAACGACC-3′) and So_putA(5′-AACAATGCTCTCTCTGTCGG-3′) from the same cDNA bank. The fragment witha size of ca. 2 kb that was obtained was cloned undirectedly in thepCR4-TOPO vector from Invitrogen (product number: 45-0030). The plasmidproduced was designated as AN 47-196. The sequence of the insertedfragments in the plasmid AN 47-196 is shown under SEQ ID NO 2.

Primers were now manufactured again in order to amplify a full-lengthsequence.

The following primers were used: SO_putA (AACAATGCTCTCTCTGTCGG) andSO_putE (CACTTTAGAAGGTATCAGAGC). A PCR product with an approximate sizeof 3.2 kb was obtained and was cloned in the pCR2.1 vector fromInvitrogen (product number: 45-0030). The plasmid obtained (filed underDSM 15926) was designated as AN 49. The sequence of the insertedfragments in the plasmid AN 49 is shown under SEQ ID NO 3.

Example 2

Information on Vectors and Plasmids

Information on Vector AN 54-196

AN 54-196 is a derivative of the plasmid pBinB33-Hyg, to which was addeda part sequence of the Class 3 branching enzyme gene as an “invertedrepeat, (RNAi technology) under the control of the promoters of thepatatin gene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989). Forthis purpose, first of all, a PCR product with the primers B1_Asp (GATGGG TAC CAG CAC TTC TAC TTG GCA GAG G) and B2_Sal (TCA AGT CGA CCA CMCCA GTC CAT TTC TGG) from a tuber-specific cDNA bank from Solanumtuberosum (cv. Désirée) was amplified, as a result of which the sitesAsp718 and SalI were added. The PCR product obtained (625 bp) was cionedin “antisense” orientation to the B33 promoter via these two sites. Asecond PCR fragment, which was amplified with the primers B3_Sal (GCTTGT CGA CGG GAG AAT TTT GTC CAG AGG) and B4_Sal (GAT CGT CGA CAG CAC TTCTAC TTG GGA GAG G) from a tuber-specific cDNA bank from Solanumtuberosum (c.v. Désirée) and which is identical to the 301 bp of thefirst fragment, was cloned via the SalI site behind the first fragment,but in “sense” orientation to the B33 promoter. This arrangement isdescribed as “inverted repeat” (RNAi technology).

Information on Vector pBinB33-Hyg

Starting from the plasmid pBinB33, the EcoRI-HindIII fragment includingthe B33 promoter, a part of the polylinker, and the ocs terminator werecut out and spliced into the correspondingly cut vector pBIB-Hyg(Becker, 1990).

The plasmid pBinB33 was obtained by splicing the promoter of the patatingene B33 from Solanum tuberosum (Rocha-Sosa et al., 1989) as a DraIfragment (nucleotide −1512-+14) into the vector pUC19 cut with SstI, theends of which had been smoothed with the help of the T4 DNA polymerase.This resulted in the plasmid pUC19-B33. The B33 promoter was cut outfrom this plasmid with EcoRI and SmaI and spliced into thecorrespondingly cut vector pBinAR. This resulted in the vegetableexpression vector pBinB33.

The plasmid pBinAR is a derivative of the vector plasmid pBin19 (Bevan,1984) and was constructed as follows:

A fragment of length 529 Bp, which included the nucleotides 6909-7437 ofthe 35S RNA promoter of the cauliflower mosaic virus (Pietrzak et al.,1986, Nucleic Acids Research 14, 5857-5868), was isolated as anEcoRI/KpnI fragment from the plasmid pDH51 (Pietrzak et al., 1986) andspliced between the EcoRI and KpnI sites of the polylinker from pUC18.This resulted in the plasmid pUC18-35S.

With the help of the restriction endonucleases HindIII und PvuII, afragment of length 192 Bp, which included the polyadenylation signal(3′-end) of the octopin synthase gene (gene 3) of the T-DNA of the Tiplasmid pTiACH5 (Gielen et al., 1984) (nucleotides 11749-11939) wasisolated from the plasmid pAGV40 (Herrera-Estrella et al., 1983). Afterthe addition of SspI linkers to the PvuII site, the fragment was splicedbetween the SphI and HindIII site from pUC18-35S. This resulted in theplasmid pA7.

The whole polylinker containing the ³⁵S promoter and the ocs terminatorwith EcoRI and HindIII was cut out of pA7 and spliced into thecorrespondingly cut pBin19. This resulted in the vegetable expressionvector pBinAR (Höfgen and Willmitzer, 1990).

Example 3

Genetically Modified Plants with Reduced Class 3 Branching EnzymeActivity

In order to produce transgenic potato plants, which have a reducedexpression of a Class 3 branching enzyme gene, the T-DNA of the plasmidAN 54-196 was transferred into potato plants of the variety Désirée withthe help of agrobacteria, as described in Rocha-Sosa et al. (EMBO J. 8,(1989), 23-29). The plants of the variety Désirée obtained bytransformation with the plasmid AN 53-196 were designated as 369SO.

Analysis with the help of non-denaturising gel electrophoresis ofprotein extracts from tubers of wild type plant cells and/or proteinextracts from genetically modified plants (396SO), showed that thegenetically modified plant cells have a reduced activity of a Class 3branching enzyme in comparison with protein extracts from tubers of wildtype plant cells.

Additionally mRNA of tuber material was extracted with standard methodsand applied to quantitative RT-PCR analysis. The analysis were performedwith a PCR-instrument ABI Prism 7700 form Applied Biosystems using theprimer St_BE-f2 (5′-TCA GGT CTA CAA GTT GAC CCG A-3′), St_BE-f2 (5′-GTAGAA CCT TCC CTT TTG TGT GA-3′) and St_BE-Fam (5′-Fam-CAT GAT CAC TCT AGCAAT CAA AGT GCC-Tamra-3′). It could be shown that given plants showedreduced transcript in comparison with the corresponding wild type.

Example 4

Potato Starch Extraction Process

All tubers of one line (0,3 to 0,7 kg) are processed jointly in acommercially available juice extractor (Multipress automatic MP80,Braun). The starch-containing fruit water is collected in a 1-l bucket(ratio bucket height: bucket diameter=approx. 1.1) containing 20 ml oftap water together with a spoon-tipful (approx. 0,3-0,4 g) of sodiumdisulphite. The bucket is subsequently filled completely with tap water.After the starch has been allowed to settle for 2 hours (h), thesupernatant is decanted off, the starch is resuspended in 1 l of tapwater and poured over a sieve with a mesh size of 125 μm. After 2 h(starch has again settled at the bottom of the bucket), the aqueoussupernatant is again decanted off. This wash step is repeated 3 moretimes so that the starch is resuspended a total of 5 times in fresh tapwater. Thereafter, the starches are dried at 37° C. to a water contentof 12-17% and homogenized using a pestle and mortar. The starches arenow available for analyses.

Example 5

Analysis of the Starch from Plants with Reduced BEIII Gene Expression

The starch from various independent lines of plants named 369SO wereisolated from potato tubers. The physico-chemical properties of thisstarch were subsequently analysed. The results of the characterizationof the modified starches are shown in the following for an example of aselection of certain plant lines. The analyses were carried out by themethods described hereinabove.

a) RVA Analysis TABLE 2 Parameters of the RVA analysis of starchisolated from wild-type plants (cv. Desiree), plants with a reducedactivity of a BEIII protein (369SO) in percent based on data of starchof the wild type. The RVA analysis was carried out as described ingeneral methods. RVA Max (%) RVA Min (%) RVA Fin (%) RVA Set (%) RVA PT(%) Gel strength cv. Desiree 100 100 100 100 100 100 369SO048 91 64 90N.d. 98 128 369SO050 84 84 89 112 98 127 369SO052 94 85 88 101 98 N.d.369SO106 91 87 89 99 98 N.d. 369SO129 87 88 93 114 99 138N.d. = not determined.

b) Analysis of the Phosphate and Amylose Content TABLE 3 Phosphate andamylose contents of starch isolated from wild-type plants (cv. Desiree),plants with a reduced activity of BEIII protein (369SO). The phosphatecontents in the C6 position of the glucose monomers and the totalphosphate content of the starch are indicated in percent based on starchfrom wild-type plants; amylose contents are indicated in percent amylosebased on the total amount of the starch, or in percent based on theamylose content of starch from wild-type_plants. Total Phosphatephosphate Amylose Amylose No. Genotype in C6 (%) in (%) (%) (% WT) 1 cv.Desiree 100.0 100.0 21.3 100.0 2 369SO048 72.8 84.8 20.8 97.7 3 369SO05079.2 78.0 20.1 94.4 4 369SO052 84.8 83.2 19.6 92.0 5 369SO106 84.8 85.920.1 94.4 6 369SO129 80.8 81.2 20.2 94.8

c) Analysis of Side-Chain Distribution The analysis of the side-chaindistribution of the amylopectin was carried out as described above. Thetable which follows is a summary of the contributions of the individualpeak areas: TABLE 5 The table shows a summary of the contributions ofthe individual peak areas of the HPAEC chromatogram in percent based onstarch from wild-type plants. Glucose cv. 369SO 369SO 369SO 369SO 369SOunits Desiree 048 050 052 106 129 dp 6 2.19 2.57 2.83 2.78 2.59 2.59 dp7 1.69 1.76 1.84 1.85 1.85 1.73 dp 8 1.35 1.34 1.37 1.38 1.44 1.36 dp 92.26 2.27 2.31 2.32 2.42 2.31 dp 10 3.74 3.81 3.86 3.94 4.00 3.85 dp 115.13 5.23 5.30 5.45 5.37 5.30 dp 12 5.99 6.14 6.18 6.32 6.17 6.17 dp 136.40 6.53 6.54 6.63 6.48 6.63 dp 14 6.39 6.45 6.44 6.49 6.37 6.52 dp 156.11 6.14 6.12 6.15 6.05 6.09 dp 16 5.74 5.75 5.72 5.75 5.68 5.72 dp 175.37 5.35 5.35 5.35 5.30 5.35 dp 18 5.08 5.04 5.06 5.05 5.01 5.06 dp 194.89 4.86 4.88 4.84 4.83 4.86 dp 20 4.68 4.59 4.65 4.60 4.60 4.62 dp 6100 117.4 129.2 126.9 118.3 118.3 dp 7 100 104.1 108.9 109.5 109.5 102.4dp 8 100 99.3 101.5 102.2 106.7 100.7 dp 9 100 100.7 102.4 102.9 107.3102.4 dp 10 100 102.0 103.3 105.5 107.1 103.1 dp 11 100 102.0 103.4106.3 104.8 103.4 dp 12 100 102.5 103.2 105.5 103.0 103.0 dp 13 100102.1 102.3 103.7 101.3 103.7 dp 14 100 100.9 100.8 101.6 99.7 102.0 dp15 100 100.5 100.2 100.7 99.0 99.7 dp 16 100 100.3 99.7 100.3 99.0 99.7dp 17 100 99.7 99.7 99.7 98.8 99.7 dp 18 100 99.3 99.7 99.5 98.7 99.7 dp19 100 99.5 99.9 99.1 98.9 99.5 dp 20 100 98.1 99.4 98.3 98.3 98.7 dp 21100 99.5 99.1 98.0 98.4 98.6 dp 22 100 98.8 99.0 97.6 98.0 98.0 dp 23100 98.8 97.7 96.2 98.3 98.8 dp 24 100 98.4 96.9 96.0 98.1 96.9 dp 25100 96.9 95.9 94.0 96.6 97.2 dp 26 100 97.1 94.2 93.5 96.4 95.6 dp 27100 95.8 93.8 91.7 95.0 95.0 dp 28 100 96.1 92.3 91.3 95.2 93.7 dp 29100 97.2 92.0 90.3 95.5 94.9 dp 30 100 94.0 91.4 89.4 93.4 93.4 dp 31100 95.6 90.8 89.2 92.4 94.0 dp 32 100 94.2 89.3 89.3 92.2 92.2 dp 33100 92.2 89.8 91.0 93.4 93.4 dp 34 100 91.9 88.9 90.4 93.3 91.9d) Analysis of the Amylopectin Side Chain Distribution by Means of GelPermeation Chromatography

Analysis of the amylopectin side chain distribution by means of gelpermeation chromatography were additionally performed.

To separate amylose and amylopectin, 100 mg of starch are dissolved in 6ml of 90% strength (v/v) DMSO with constant stirring. After addition of3 volumes of ethanol, the precipitate is separated off by centrifugationfor 10 minutes at 1800×g at room temperature. The pellet is subsequentlywashed with 30 ml of ethanol, dried and dissolved in 10 ml of 1%strength (w/v) NaCl solution at 60° C. After cooling the solution to 30°C., approximately 50 mg of thymol are added slowly, and this solution isincubated for 2 to 3 days at 30° C. The solution is subsequentlycentrifuged for 30 minutes at 2000×g at room temperature. Thesupernatant is treated with three volumes of ethanol, and theamylopectin which precipitates is separated off by centrifugation for 5minutes at 2000×g at room temperature. The pellet (amylopectin) iswashed with 10 ml of 70% strength (v/v) ethanol, centrifuged for 10minutes at 2000×g at room temperature and then dried using acetone.

10 mg of amylopectin are subsequently stirred for 10 minutes at 70° C.in 250 μl of 90% strength (v/v) DMSO. 375 μl of water at a temperatureof 80° C. are added to the solution until dissolution is complete.

200 μl of this solution are treated with 300 μl of a 16.6 mM sodiumacetate solution pH 3.5 and 2 μl of isoamylase (0.24 u/μl, Megazyme,Sydney, Australia) and the mixture is incubated for 15 hours at 37° C.

A 1:4 dilution of this aqueous isoamylase reaction mixture with DMSO,comprising 90 mM sodium nitrate, is subsequently filtered through a 0.2μm filter, and 24 μl of the filtrate is analysed chromatographically.Separation was carried out with two columns connected in series, first aGram PSS3000 (Polymer Standards Service, with suitable precolumn),followed by a Gram PSS100. Detection was by means of refraction indexdetector (RI 71, Shodex). The column was equilibrated with DMSOcomprising 90 mM sodium nitrate. It was eluted with DMSO comprising 90mM sodium nitrate at a flow rate of 0.7 ml/min over a period of 1 hour.

To correlate the elution volume with the molecular mass, the column usedwas calibrated with dextran standards. The dextrans used, theirmolecular mass and the elution volumes are shown in Table 6. Using theresulting calibration graph, the elution diagram was pictured as amolecular weight distribution.

The chromatograms obtained were further evaluated using the programWingpc Version 6 from Polymer Standards Service GmbH, Mainz, Germany.

The total area under the line of the GPC chromatogram was divided intoindividual segments, each of which represent groups of side chains ofdifferent lengths. The chosen segments contained glucan chains with thefollowing degree of polymerization (DP=number of glucose monomers withinone side chain): DP<12, DP12-18, DP19-24, DP25-30, DP31-36, DP37-42,DP43-48, DP49-55, DP56-61 and DP62-123. To determine the molecularweight of the individual side chains, a molecular weight of 162 wasassumed for glucose. The total area under the line in the GPCchromatogram was then set as 100%, and the percentage of the areas ofthe individual segments was calculated based on the percentage of thetotal area. Results obtained from this analysis are shown in Table 7.TABLE 6 Calibration table. elution volume [ml] molar mass [D] sample18.76 401300 Dextran T670 19.41 276500 Dextran T410 20.49 196300 DextranT270 21.35 123600 Dextran T150 22.45 66700 Dextran T80 23.52 43500Dextran T50 25.15 21400 Dextran T25 26.92 9890 Dextran T12 28.38 4440Dextran T5 30.77 1080 Dextran T1

TABLE 7 Side chain profiles DP < 12, DP 12 to 18, DP 19 to 24, DP 25 to30, DP 31 to 36, DP 37 to 42, DP 43-48, DP 49 to 55, DP 56 to 61 and DP62 to 123 for amylopectin isolated from wild-type plants (cv. Desiree)and from plants with a reduced activity of a BEIII protein (369SO).degree of % total area polymerisation cv. Desiree 369 SO 48 369 SO 50369 SO 52 369 SO 106 369 SO 129 <dp12 16.49 16.57 17.07 17.73 17.5917.64 dp12-19 13.89 14.47 14.22 14.82 14.16 14.23 dp20-25 15.74 16.5416.15 16.74 16.32 16.51 dp26-31 9.41 9.73 9.57 9.80 9.86 9.88 dp32-378.53 8.53 8.45 8.56 8.59 8.45 dp38-43 6.82 6.67 6.57 6.63 6.58 6.44dp44-49 6.05 5.91 5.81 5.83 5.79 5.72 dp50-56 4.88 4.78 4.71 4.66 4.704.67 dp57-62 4.26 4.15 4.10 3.98 4.09 4.10 dp63-123 13.92 12.66 13.3511.25 12.31 12.38

TABLE 8 Side chain profiles DP < 12, DP 12 to 18, DP 19 to 24, DP 25 to30, DP 31 to 36, DP 37 to 42, DP 43-48, DP 49 to 55, DP 56 to 61 and DP62 to 123 for amylopectin isolated from wild-type plants (cv. Desiree)and from plants with a reduced activity of a BEIII protein (369SO). Thepercentages indicate the modification of the individual side chainprofiles based on amylopectin isolated from wild-type plants. degree of% WT polymerisation cv. Desiree 369 SO 48 369 SO 50 369 SO 52 369 SO 106369 SO 129 <dp12 100.00 100.47 103.53 107.53 106.69 106.96 dp12-19100.00 104.13 102.37 106.68 101.90 102.40 dp20-25 100.00 105.11 102.62106.32 103.69 104.87 dp26-31 100.00 103.34 101.67 104.09 104.81 104.92dp32-37 100.00 100.01 99.05 100.38 100.73 99.04 dp38-43 100.00 97.7496.40 97.23 96.54 94.48 dp44-49 100.00 97.68 96.00 96.27 95.76 94.48dp50-56 100.00 97.90 96.45 95.57 96.26 95.78 dp57-62 100.00 97.36 96.0793.44 95.91 96.13 dp63-123 100.00 90.95 95.89 80.82 88.41 88.89e) DSC-Analysis (“Differential Scanning Calorimetry”)

Investigations with the aid of DSC-analysis (“Differential ScanningCalorimetry”) have been done by the method described by WO 01/19975.Results obtained from this analysis are shown in Table 9. TABLE 9Parameters of the DSC analysis of starch isolated from wild-type plants(cv. Desiree), plants with a reduced activity of a BEIII protein (369SO)indicated in ° C. respectively J/g and in percent based on data ofstarch of the wild type. The DSC analysis was carried out as describedin general methods. T0 [° C.] = peak onset, T Peak [° C.] = Peaktemperature, dH [J/g] = heat of_melting. TO (° C.) TO (%) T Peak (° C.)T Peak (%) dH (J/g) dH (J/g) cv. Desiree 64.84 100 68.09 100 20.31 100369SO048 64.32 99.2 67.16 98.6 20.33 100.1 369SO050 63.35 97.7 66.7598.0 20.63 101.6 369SO052 63.27 97.6 66.46 97.6 21.23 104.5 369SO10663.77 98.3 66.96 98.3 21.42 105.5 369SO129 63.75 98.3 67.41 99.0 20.57101.3

1. A genetically modified plant cell having a reduced activity of atleast one Class 3 branching enzyme in comparison with corresponding wildtype plant cells that have not been genetically modified.
 2. Thegenetically modified plant cell according to claim 1, wherein thegenetically modified plant cell comprises at least one foreign nucleicacid molecule that has been introduced into the genome of the plantcell.
 3. The genetically modified plant cell according to claim 2,wherein the foreign nucleic acid molecule codes a Class 3 branchingenzyme.
 4. The genetically modified plant cell according to claim 3,wherein said foreign nucleic acid molecule is a) a nucleic acid moleculemolecules, which codes a protein with the amino acid sequence of Seq IDNo. 4; b) a nucleic acid molecule molecules, which codes a protein, theamino acid sequence of which has an identity of at least 50% with theamino acid sequence of SEQ ID NO: 4; c) a nucleic acid molecule, whichincludes the nucleotide sequence of Seq ID No. 3 or a complementarysequence; d) a nucleic acid molecule, the nucleic acid sequence of whichhas an identity of at least 50% with the nucleic acid sequencesdescribed under a) or c); e) a nucleic acid molecule, which hybridizeswith at least one strand of the nucleic acid molecules described undera) or c) under stringent conditions; f) a nucleic acid molecule, thenucleotide sequence of which deviates from the sequence of the nucleicacid molecules identified under a), b), c), d), e) or f) due to thedegeneration of the genetic code; or g) a nucleic acid molecule, whichrepresents fragments, allelic variants and/or derivatives of the nucleicacid molecules identified under a), b), c), d), e) or f).
 5. Thegenetically modified plant cell according to claim 2, wherein saidforeign nucleic acid molecule is a) a DNA molecule, which codes at leastone antisense RNA, which effects a reduction in the expression of atleast one endogenous gene, which codes a Class 3 branching enzyme; b) aDNA molecule, which by means of a co-suppression effect leads to thereduction in the expression of at least one endogenous gene, which codesa Class 3 branching enzyme; c) a DNA molecule, which codes at least oneribozyme, which splits specific transcripts of at least one endogenousgene, which codes a Class 3 branching enzyme; d) a DNA molecule, whichsimultaneously codes at least one antisense RNA and at least one senseRNA, wherein said antisense RNA and said sense RNA form adouble-stranded RNA molecule, which effects a reduction in theexpression of at least one endogenous gene, which codes a Class 3branching enzyme; e) a nucleic acid molecule introduced by means of invivo mutagenesis, which leads to a mutation or an insertion of aheterologous sequence in at least one endogenous gene coding a Class 3branching enzyme, wherein the mutation or insertion effects a reductionin the expression of a gene coding a Class 3 branching enzyme or resultsin the synthesis of inactive Class 3 branching enzymes; f) a nucleicacid molecule, which codes an antibody, wherein the antibody results ina reduction in the activity of a Class 3 branching enzyme due to thebonding to a Class 3 branching enzyme; g) a DNA molecule, which containstransposons, wherein the integration of said transposons leads to amutation or an insertion in at least one endogenous gene coding a Class3 branching enzyme, which effects a reduction in the expression of atleast one gene coding a Class 3 branching enzyme, or results in thesynthesis of inactive Class 3 branching enzymes; or h) a T-DNA molecule,which, due to insertion in at least one endogenous gene coding a Class 3branching enzyme, effects a reduction in the expression of at least onegene coding a Class 3 branching enzyme, or result in the synthesis ofinactive Class 3 branching enzyme.
 6. A plant cell according to claim 1,which synthesizes a modified starch in comparison with correspondingwild type plant cells that have not been genetically modified.
 7. Aplant containing plant cells according to claim
 1. 8. The plantaccording to claim 7, which is a starch-storing plant.
 9. The plantaccording to claim 8, which is a maize, rice, wheat, rye, oat, barley,cassaya, potato, sago, mung bean, pea or sorghum plant.
 10. The plantaccording to claim 8, which is a potato plant.
 11. Propagation materialof plants according to claim
 7. 12. Harvestable plant parts of plantsaccording to claim
 7. 13. A method for the manufacture of a geneticallymodified plant according to claim 7, comprising; a) geneticallymodifying a plant cell, whereby the genetic modification leads to thereduction of the activity of a Class 3 vegetable branching enzyme incomparison with corresponding wild type plant cells that have not beengenetically modified; b) regenerating a plant from plant cells from Stepa); and c) if necessary, producing further plants with the help of theplants according to Step b).
 14. The method according to claim 13,wherein the genetic modification comprises introduction of at least oneforeign nucleic acid molecule into the genome of the plant.
 15. Themethod according to claim 14, wherein the said foreign nucleic acidmolecule is a) a nucleic acid molecule, which codes a protein with theamino acid sequence of Seq ID No. 4; b) a nucleic acid molecule, whichcodes a protein, the amino acid sequence of which has an identity of atleast 50% with the amino acid sequence of SEQ ID NO: 4; c) a nucleicacid molecule, which include includes the nucleotide sequence shownunder of Seq ID No. 3 or a complimentary complementary sequence; d) anucleic acid molecule, the nucleic acid sequence of which has anidentity of at least 50% with the nucleic acid sequences described undera) or c); e) a nucleic acid molecule molecules, which hybridisehybridizes with at least one strand of the nucleic acid moleculesdescribed under a) or c) under stringent conditions; f) a nucleic acidmolecule, the nucleotide sequence of which deviates from the sequence ofthe nucleic acid molecules identified under a), b), c), d), e) or f) dueto the degeneration of the genetic code; or g) a nucleic acid molecule,which represents fragments, allelic variants and/or derivatives of thenucleic acid molecules identified under a), b), c), d), e) or f). 16.The method according to claim 14, wherein said foreign nucleic acidmolecule is a) a DNA molecule, which codes at least one antisense RNA,which effects a reduction in the expression of at least one endogenousgene, which codes a Class 3 branching enzyme; b) a DNA molecule, whichby means of a co-suppression effect leads to the reduction in theexpression of at least one endogenous gene, which codes a Class 3branching enzyme; c) a DNA molecule, which codes at least one ribozyme,which splits specific transcripts of at least one endogenous gene, whichcodes a Class 3 branching enzyme; d) a DNA molecule, whichsimultaneously codes at least one antisense RNA and at least one senseRNA, wherein said antisense RNA and the said sense RNA form adouble-stranded RNA molecule, which effects a reduction in theexpression of at least one endogenous gene, which codes a Class 3branching enzyme; e) a nucleic acid molecule introduced by means of invivo mutagenesis, which lead to a mutation or an insertion of aheterologous sequence in at least one endogenous gene coding a Class 3branching enzyme, wherein the mutation or insertion effects a reductionin the expression of a gene coding a Class 3 branching enzyme or resultsin the synthesis of inactive Class 3 branching enzymes; f) a nucleicacid molecule, which codes an antibody, wherein the antibody results ina reduction in the activity of a Class 3 branching enzyme due to thebonding to a Class 3 branching enzyme; g) a DNA molecule, which containstransposons, wherein the integration of said transposons leads to amutation or an insertion in at least one endogenous gene coding a Class3 branching enzyme, which effects a reduction in the expression of atleast one gene coding a Class 3 branching enzyme, or results in thesynthesis of inactive Class 3 branching enzymes; or h) a T-DNA molecule,which, due to insertion in at least one endogenous gene coding a Class 3branching enzyme, effects a reduction in the expression of at least onegene coding a Class 3 branching enzyme, or result in the synthesis ofinactive Class 3 branching enzyme.
 17. The method according to claim 13,wherein the genetically modified plant synthesizes a modified starch incomparison with corresponding wild type plants that have not beengenetically modified.
 18. A nucleic acid molecule, coding for a proteinwith the enzymatic activity of a Class 3 branching enzyme, comprising a)a nucleic acid molecule, which codes a protein with the amino acidsequence of Seq ID No. 4; b) a nucleic acid molecule, which codes aprotein, the amino acid sequence of which has an identity of at least70% with the amino acid sequence of SEQ ID NO: 4; c) a nucleic acidmolecule, which includes the nucleotide sequence of Seq ID No. 3 or acomplimentary sequence; d) a nucleic acid molecule, which has anidentity of at least 70% with the nucleic acid sequences described undera) or c); e) a nucleic acid molecule, which hybridizes with at least onestrand of the nucleic acid molecules described under a) or c) understringent conditions; f) a nucleic acid molecule, the nucleotidesequence of which deviates from the sequence of the nucleic acidmolecules identified under a), b), c), d), e) or f) due to thedegeneration of the genetic code; or g) a nucleic acid molecule, whichrepresents fragments, allelic variants and/or derivatives of the nucleicacid molecules identified under a), b), c), d), e) or f).
 19. Thenucleic acid molecule according to claim 18, which codes a Class 3branching enzyme of potato.
 20. A vector comprising a nucleic acidmolecule according to claim
 18. 21. The vector according to claim 20,wherein the nucleic acid molecule is linked with regulatory sequencesfor transcription into prokaryotic or eukaryotic cells.
 22. A vectorcomprising a foreign nucleic acid molecule defined as in claim 5 undera), b), c) or d).
 23. A host cell, which is genetically modified with anucleic acid molecule according to claim 18 or with a vector accordingto claim
 20. 24. A protein with the enzymatic activity of a Class 3branching enzyme, comprising a) a protein, which includes the amino acidsequence specified under of SEQ ID No. 4, or b) a protein, which has anidentity of at least 70% with the amino acid sequence of the proteinsidentified under a).
 25. The protein according to claim 24, wherein theClass 3 branching enzyme comes from a potato plant.
 26. A modifiedstarch obtainable from a genetically modified plant according to claim7, from propagation material according to claim 11, or from harvestableplant parts according to claim
 12. 27. A method for the manufacture of amodified starch including the step of extracting the starch from a plantcell according to claim
 1. 28. A method for the manufacture of amodified starch including the step of extracting the starch from a plantaccording to claim 7, or from starch-storing parts of such a plant. 29.A method for the manufacture of a modified starch including the step ofextracting the starch from harvestable plant parts according to claim12.
 30. A method for the manufacture of a derived starch, whereinmodified starch according to claim 26 or obtainable by the methodaccording to claim 27 is derived.
 31. (canceled)
 32. A modified starchobtainable by the method according to claim
 27. 33. Derived starchobtainable by the method according to claim
 30. 34. (canceled)